Label-free high-throughput optical technique for detecting biomolecular interactions

ABSTRACT

Methods and compositions are provided for detecting biomolecular interactions. The use of labels is not required and the methods can be performed in a high-throughput manner. The invention also provides optical devices useful as narrow band filters.

PRIORITY

[0001] This application claims the benefit of U.S. provisionalapplication 60/244,312 filed Oct. 30, 2000; U.S. provisional application60/283,314 filed Apr. 12, 2001; and U.S. provisional application60/303,028 filed Jul. 3, 2001.

TECHNICAL AREA OF THE INVENTION

[0002] The invention relates to compositions and methods for detectingbiomolecular interactions. The detection can occur without the use oflabels and can be done in a high-throughput manner. The invention alsorelates to optical devices.

BACKGROUND OF THE INVENTION

[0003] With the completion of the sequencing of the human genome, one ofthe next grand challenges of molecular biology will be to understand howthe many protein targets encoded by DNA interact with other proteins,small molecule pharmaceutical candidates, and a large host of enzymesand inhibitors. See e.g., Pandey & Mann, “Proteomics to study genes andgenomes,” Nature, 405, p. 837-846, 2000; Leigh Anderson et al.,“Proteomics: applications in basic and applied biology,” Current Opinionin Biotechnology, 11, p. 408-412, 2000; Patterson, “Proteomics: theindustrialization of protein chemistry,” Current Opinion inBiotechnology, 11, p. 413-418, 2000; MacBeath & Schreiber, “PrintingProteins as Microarrays for High-Throughput Function Determination,”Science, 289, p. 1760-1763, 2000; De Wildt et al., “Antibody arrays forhigh-throughput screening of antibody-antigen interactions,” NatureBiotechnology, 18, p. 989-994, 2000. To this end, tools that have theability to simultaneously quantify many different biomolecularinteractions with high sensitivity will find application inpharmaceutical discovery, proteomics, and diagnostics. Further, forthese tools to find widespread use, they must be simple to use,inexpensive to own and operate, and applicable to a wide range ofanalytes that can include, for example, polynucleotides, peptides, smallproteins, antibodies, and even entire cells.

[0004] Biosensors have been developed to detect a variety ofbiomolecular complexes including oligonucleotides, antibody-antigeninteractions, hormone-receptor interactions, and enzyme-substrateinteractions. In general, biosensors consist of two components: a highlyspecific recognition element and a transducer that converts themolecular recognition event into a quantifiable signal. Signaltransduction has been accomplished by many methods, includingfluorescence, interferometry (Jenison et al., “Interference-baseddetection of nucleic acid targets on optically coated silicon,” NatureBiotechnology, 19, p. 62-65; Lin et al., “A porous silicon-based opticalinterferometric biosensor,” Science, 278, p. 840-843, 1997), andgravimetry (A. Cunningham, Bioanalytical Sensors, John Wiley & Sons(1998)).

[0005] Of the optically-based transduction methods, direct methods thatdo not require labeling of analytes with fluorescent compounds are ofinterest due to the relative assay simplicity and ability to study theinteraction of small molecules and proteins that are not readilylabeled. Direct optical methods include surface plasmon resonance (SPR)(Jordan & Corn, “Surface Plasmon Resonance Imaging Measurements ofElectrostatic Biopolymer Adsorption onto Chemically Modified GoldSurfaces,” Anal. Chem., 69:1449-1456 (1997), (grating couplers (Morhardet al., “Immobilization of antibodies in micropatterns for celldetection by optical diffraction,” Sensors and Actuators B, 70, p.232-242, 2000), ellipsometry (Jin et al., “A biosensor concept based onimaging ellipsometry for visualization of biomolecular interactions,”Analytical Biochemistry, 232, p. 69-72, 1995), evanascent wave devices(Huber et al., “Direct optical immunosensing (sensitivity andselectivity),” Sensors and Actuators B, 6, p. 122-126, 1992), andreflectometry (Brecht & Gauglitz, “Optical probes and transducers,”Biosensors and Bioelectronics, 10, p. 923-936, 1995). Theoreticallypredicted detection limits of these detection methods have beendetermined and experimentally confirmed to be feasible down todiagnostically relevant concentration ranges. However, to date, thesemethods have yet to yield commercially available high-throughputinstruments that can perform high sensitivity assays without any type oflabel in a format that is readily compatible with the microtiterplate-based or microarray-based infrastructure that is most often usedfor high-throughput biomolecular interaction analysis. Therefore, thereis a need in the art for compositions and methods that can achieve thesegoals.

SUMMARY OF THE INVENTION

[0006] It is an object of the invention to provide compositions andmethods for detecting binding of one or more specific binding substancesto their respective binding partners. This and other objects of theinvention are provided by one or more of the embodiments describedbelow.

[0007] One embodiment of the invention provides a biosensor comprising:a two-dimensional grating comprised of a material having a highrefractive index, a substrate layer that supports the two-dimensionalgrating, and one or more specific binding substances immobilized on thesurface of the two-dimensional grating opposite of the substrate layer.When the biosensor is illuminated a resonant grating effect is producedon the reflected radiation spectrum. The depth and period of thetwo-dimensional grating are less than the wavelength of the resonantgrating effect.

[0008] Another embodiment of the invention provides an optical devicecomprising a two-dimensional grating comprised of a material having ahigh refractive index and a substrate layer that supports thetwo-dimensional grating. When the optical device is illuminated aresonant grating effect is produced on the reflected radiation spectrum.The depth and period of the two-dimensional grating are less than thewavelength of the resonant grating effect.

[0009] A narrow band of optical wavelengths can be reflected from thebiosensor or optical device when the biosensor is illuminated with abroad band of optical wavelengths. The substrate can comprise glass,plastic or epoxy. The two-dimensional grating can comprise a materialselected from the group consisting of zinc sulfide, titanium dioxide,tantalum oxide, and silicon nitride.

[0010] The substrate and two-dimensional grating can optionally comprisea single unit. The surface of the single unit comprising thetwo-dimensional grating is coated with a material having a highrefractive index, and the one or more specific binding substances areimmobilized on the surface of the material having a high refractiveindex opposite of the single unit. The single unit can be comprised of amaterial selected from the group consisting of glass, plastic, andepoxy.

[0011] The biosensor or optical device can optionally comprise a coverlayer on the surface of the two-dimensional grating opposite of thesubstrate layer. The one or more specific binding substances areimmobilized on the surface of the cover layer opposite of thetwo-dimensional grating. The cover layer can comprise a material thathas a lower refractive index than the high refractive index material ofthe two-dimensional grating. For example, a cover layer can compriseglass, epoxy, and plastic.

[0012] A two-dimensional grating can be comprised of a repeating patternof shapes selected from the group consisting of lines, squares, circles,ellipses, triangles, trapezoids, sinusoidal waves, ovals, rectangles,and hexagons. The repeating pattern of shapes can be arranged in alinear grid, i.e., a grid of parallel lines, a rectangular grid, or ahexagonal grid. The two-dimensional grating can have a period of about0.01 microns to about 1 micron and a depth of about 0.01 microns toabout 1 micron.

[0013] The one or more specific binding substances can be arranged in anarray of distinct locations and can be immobilized on thetwo-dimensional grating by physical adsorption or by chemical binding.The distinct locations can define a microarray spot of about 50-500 or150-200 microns in diameter. The one or more specific binding substancescan be bound to their binding partners. The one or more specific bindingsubstances can be selected from the group consisting of nucleic acids,polypeptides, antigens, polyclonal antibodies, monoclonal antibodies,single chain antibodies (scFv), F(ab) fragments, F(ab′)₂ fragments, Fvfragments, small organic molecules, cells, viruses, bacteria, andbiological samples. The biological sample can be selected from the groupconsisting of blood, plasma, serum, gastrointestinal secretions,homogenates of tissues or tumors, synovial fluid, feces, saliva, sputum,cyst fluid, amniotic fluid, cerebrospinal fluid, peritoneal fluid, lunglavage fluid, semen, lymphatic fluid, tears, and prostatitc fluid. Thebinding partners can be selected from the group consisting of nucleicacids, polypeptides, antigens, polyclonal antibodies, monoclonalantibodies, single chain antibodies (scFv), F(ab) fragments, F(ab′)₂fragments, Fv fragments, small organic molecules, cells, viruses,bacteria, and biological samples. The biosensor can further comprise anantireflective dielectric coating on a surface of the substrate oppositeof the two-dimensional grating. The biosensor can comprise anantireflective physical structure that is embossed into a surface of thesubstrate opposite of the two-dimensional grating, such as a motheyestructure. The biosensor can comprise an internal surface of aliquid-containing vessel. The vessel is selected from the groupconsisting of a microtiter plate, a test tube, a petri dish and amicrofluidic channel. The biosensor can be attached to a bottomlessmicrotiter plate by a method selected from the group consisting ofadhesive attachment, ultrasonic welding and laser welding.

[0014] Another embodiment of the invention provides a detection systemcomprising a biosensor or optical device of the invention, a lightsource that directs light to the biosensor or optical device, and adetector that detects light reflected from the biosensor. The detectionsystem can comprise a fiber probe comprising one or more illuminatingoptical fibers that are connected at a first end to the light source,and one or more collecting optical fibers connected at a first end tothe detector, wherein the second ends of the illuminating and collectingfibers are arranged in line with a collimating lens that focuses lightonto the biosensor or optical device. The illuminating fiber and thecollecting fiber can be the same fiber. The light source can illuminatethe biosensor from its top surface or from its bottom surface.

[0015] Even another embodiment of the invention provides a method ofdetecting the binding of one or more specific binding substances totheir respective binding partners. The method comprises applying one ormore binding partners to a biosensor of the invention, illuminating thebiosensor with light, and detecting a maxima in reflected wavelength, ora minima in transmitted wavelength of light from the biosensor. Whereone or more specific binding substances have bound to their respectivebinding partners, the reflected wavelength of light is shifted.

[0016] Still another embodiment of the invention provides a method ofdetecting the binding of one or more specific binding substances totheir respective binding partners. The method comprises applying one ormore binding partners to a biosensor of the invention, wherein thebiosensor comprises a two-dimensional grating that is coated with anarray of distinct locations containing the one or more specific bindingsubstances. Each distinct location of the biosensor is illuminated withlight, and maximum reflected wavelength or minimum transmittedwavelength of light is detected from each distinct location of thebiosensor. Where the one or more specific binding substances have boundto their respective binding partners at a distinct location, thereflected wavelength of light is shifted.

[0017] Yet another embodiment of the invention provides a method ofdetecting activity of an enzyme. The method comprises applying one ormore enzymes to a biosensor of the invention, washing the biosensor,illuminating the biosensor with light, and detecting reflectedwavelength of light from the biosensor. Where the one or more enzymeshave altered the one or more specific binding substances of thebiosensor by enzymatic activity, the reflected wavelength of light isshifted.

[0018] Another embodiment of the invention provides a biosensorcomprising a sheet material having a first and second surface, whereinthe first surface defines relief volume diffraction structures, areflective material coated onto the first surface of the sheet material,and one or more specific binding substances immobilized on thereflective material. Still another embodiment of the invention providesan optical device comprising a sheet material having a first and secondsurface, wherein the first surface defines relief volume diffractionstructures, and a reflective material coated onto the first surface ofthe sheet material. The biosensor or optical device reflects lightpredominantly at a first single optical wavelength when illuminated witha broad band of optical wavelengths. The biosensor reflects light at asecond single optical wavelength when the one or more specific bindingsubstances are immobilized on the reflective surface. The reflection atthe first and second optical wavelengths results from opticalinterference. The biosensor can reflect light at a third single opticalwavelength when the one or more specific binding substances are bound totheir respective binding partners. The reflection at the third opticalwavelength results from optical interference. The depth and period ofthe relief volume diffraction structures can be less than the resonancewavelength of the light reflected from the biosensor. The depth of therelief volume diffraction structures can be about 0.01 microns to about1 micron. The period of the relief volume diffraction structures can beabout 0.01 microns to about 1 micron. The relief volume diffractionstructures can be about 0.5 microns to about 5 microns in diameter.

[0019] Even another embodiment of the invention provides a biosensorcomprising a two-dimensional grating having a first and a second surfacecomprised of an optically transparent material that conductselectricity. The first surface of the grating is coated with anelectrical insulator, and the second surface of the grating is depositedon a substrate. When the biosensor is illuminated, a resonant gratingeffect is produced on the reflected radiation spectrum. The depth andthe period of the grating are less than the wavelength of the resonantgrating effect. Two or more separate grating regions can be present onthe same substrate. An electrically conducting trace to each separategrating region of the substrate can be present. The conducting trace canbe connected to a voltage source. One or more specific bindingsubstances can be bound to each separate grating region of thesubstrate.

[0020] Yet another embodiment of the invention provides a method ofmeasuring the amount of binding partners in a test sample. One or morebinding partners are immobilized to the biosensor described above. Anelectrical charge is applied to the electrically conducting traces. Thebiosensor is illuminated with light and the reflected wavelength oflight is detected from the biosensor. Where the one or more specificbinding substances have bound to their respective binding partners, thereflected wavelength of light is shifted. A reversed electrical chargecan be applied to the electrically conducting traces before illuminatingthe biosensor with light.

[0021] Still another embodiment of the invention provides a method ofdetecting the binding of one or more specific binding substances totheir respective binding partners. The method comprises illuminating abiosensor of the invention with light, detecting reflected wavelength oflight from the biosensor, applying a test sample comprising one or morebinding partners to the biosensor, illuminating the biosensor withlight, and detecting reflected wavelength of light from the biosensor.The difference in wavelength of light is a measurement of the amount ofone or more binding partners in the test sample.

[0022] Another embodiment of the invention provides a detection systemcomprising a biosensor of the invention, a light source that directslight at the biosensor, and a detector that detects light reflected fromthe biosensor. A first illuminating fiber probe having two ends isconnected at its first end to the detector. A second collection fiberprobe having two ends is connected at its first end to the light source.The first and second fiber probes are connected at their second ends toa third fiber probe, which acts as an illumination and collection fiberprobe. The third fiber probe is oriented at a normal angle of incidenceto the biosensor and supports counter-propagating illuminating andreflecting optical signals.

[0023] Even another embodiment of the invention provides a detectionsystem comprising a biosensor of the invention, a light source thatdirects light at the biosensor, and a detector that detects lightreflected from the biosensor. An illuminating fiber probe is connectedto the light source and is oriented at a 90 degree angle to a collectingfiber probe. The collecting fiber probe is connected to the detector,wherein light is directed through the illuminating fiber probe into abeam splitter that directs the light to the biosensor. Reflected lightis directed into the beam splitter that directs the light into thecollecting fiber.

[0024] Still another embodiment of the invention comprises a method ofimmobilizing one or more specific binding substances onto a biosensor ofthe invention. The method comprises activating the biosensor with amine,attaching linker groups to the amine-activated biosensor, and attachingone or more specific binding substances to the linker groups. Thebiosensor can be activated with amine by a method comprising immersingthe biosensor into a piranha solution, washing the biosensor, immersingthe biosensor in 3% 3-aminopropyltriethoxysilane solution in dryacetone, washing the biosensor in dry acetone, and washing the biosensorwith water. A linker can be selected from the group consisting of amine,aldehyde, N,N′-disuccinimidyl carbonate, and nickel.

[0025] Yet another embodiment of the invention provides a method ofdetecting the binding of one or more specific binding substances totheir respective binding partners. The method comprises applying one ormore binding partners comprising one or more tags to a biosensor of theinvention, illuminating the biosensor with light, and detectingreflected wavelength of light from the biosensor. Where the one or morespecific binding substances have bound to their respective bindingpartners, the reflected wavelength of light is shifted. The one or moretags can be selected from the group consisting of biotin, SMPT, DMP,NNDC, and histidine. The one or more tags can be reacted with acomposition selected from the group consisting of streptavidin,horseradish peroxidase, and streptavidin coated nanoparticles, beforethe step of illuminating the biosensor with light.

[0026] Another embodiment of the invention provides a biosensorcomposition comprising two or more biosensors of the invention, wherethe biosensors are associated with a holding fixture. The biosensorcomposition can comprise about 96, about 384, or about 50 to about 1,000individual biosensors. Each of the two or more biosensors can compriseabout 25 to about 1,000 distinct locations. Each biosensor can be about1 mm² to about 5 mm², or about 3 mm². The holding fixture can hold eachbiosensor such that each biosensor can be placed into a separate well ofa microtiter plate.

[0027] Even another embodiment of the invention provides a biosensorcomposition comprising one or more biosensors of the invention on a tipof a multi-fiber optic probe. The one or more biosensors can befabricated into the tip of the probe or can be attached onto the tip ofthe probe.

[0028] Still another embodiment of the invention provides a method ofdetecting binding of one or more specific binding substances to theirrespective binding partners in vivo. The method comprises inserting thetip of the fiber optic probe described above into the body of a human oranimal, illuminating the biosensor with light, and detecting reflectedwavelength of light from the biosensor. If the one or more specificbinding substances have bound to their respective binding partners, thenthe reflected wavelength of light is shifted.

[0029] Yet another embodiment of the invention provides a detectionsystem comprising a biosensor of the invention, a laser source thatdirects a laser beam to a scanning mirror device, wherein the scanningmirror device is used to vary the laser beam's incident angle, anoptical system for maintaining columination of the incident laser beam,and a light detector. The scanning mirror device can be a lineargalvanometer. The linear galvanometer can operate at a frequency ofabout 2 Hz to about 120 Hz and a mechanical scan angle of about 10degrees to about 20 degrees. The laser can be a diode laser with awavelength selected from the group consisting of 780 nm, 785 nm, 810 nm,and 830 nm.

[0030] Another embodiment of the invention provides a method fordetermining a location of a resonant peak for a binding partner in aresonant reflectance spectrum with a colormetric resonant biosensor. Themethod comprises selecting a set of resonant reflectance data for aplurality of colormetric resonant biosensor distinct locations. The setof resonant reflectance data is collected by illuminating a colormetricresonant diffractive grating surface with a light source and measuringreflected light at a predetermined incidence. The colormetric resonantdiffractive grating surface is used as a surface binding platform forone or more specific binding substances and binding partners can bedetected without the use of a molecular label. The set of resonantreflectance data includes a plurality of sets of two measurements, wherea first measurement includes a first reflectance spectra of one or morespecific binding substances that are attached to the colormetricresonant diffractive grating surface and a second measurement includes asecond reflectance spectra of the one or more specific bindingsubstances after one or more binding partners are applied to colormetricresonant diffractive grating surface including the one or more specificbinding substances. A difference in a peak wavelength between the firstand second measurement is a measurement of an amount of binding partnersthat bound to the one or more specific binding substances. A maximumvalue for a second measurement from the plurality of sets of twomeasurements from the set of resonant reflectance data for the pluralityof binding partners is determined, wherein the maximum value includesinherent noise included in the resonant reflectance data. Whether themaximum value is greater than a pre-determined threshold is determined,and if so, a curve-fit region around the determined maximum value isdetermined and a curve-fitting procedure is performed to fit a curvearound the curve-fit region, wherein the curve-fitting procedure removesa predetermined amount of inherent noise included in the resonantreflectance data. A location of a maximum resonant peak on the fittedcurve is determined. A value of the maximum resonant peak is determined,wherein the value of the maximum resonant peak is used to identify anamount of biomolecular binding of the one or more specific bindingsubstances to the one or more binding partners.

[0031] The sensitivity of a colormetric resonant biosensor can bedetermined by a shift in a location of a resonant peak in the pluralityof sets of two measurements in the set of resonant reflectance data. Thestep of selecting a set of resonant reflectance data can includeselecting a set of resonant reflectance data:

x _(i)andy _(i)fori=1, 2, 3, . . . n,

[0032] wherein x_(i) is where a first measurement includes a firstreflectance spectra of one or more specific binding substance attachedto the colormetric resonant diffractive grating surface, y_(i) a secondmeasurement includes a second reflectance spectra of the one or morespecific binding substances after a plurality of binding partners areapplied to colormetric resonant diffractive grating surface includingthe one or more specific binding substances, and n is a total number ofmeasurements collected. The step of determining a maximum value for asecond measurement can include determining a maximum value y_(k) suchthat:

(y _(k) >=y _(i)) for all i≠k.

[0033] The step of determining whether the maximum value is greater thana pre-determined threshold can include computing a mean of the set ofresonant reflectance data, computing a standard deviation of the set ofresonant reflectance data, and determining whether((y_(k)−mean)/standard deviation) is greater than a pre-determinedthreshold. The step of defining a curve-fit region around the determinedmaximum value can include defining a curve-fit region of (2w+1) bins,wherein w is a pre-determined accuracy value, extracting (x_(i),k−w<=i<=k+w), and extracting (y_(i), k−w<=i<=k+w). The step ofperforming a curve-fitting procedure can include computing g_(i)=Iny_(i), performing a 2^(nd)nd order polynomial fit on g_(i) to obtaing′_(i) defined on (x₁, k−w<=i<=k+w), determining from the 2^(nd) orderpolynomial fit coefficients a, b and c of for (ax²+bx+c)−, and computingy′_(i)=e^(g′i). The step of determining a location of a maximum resonantpeak on the fitted curve can include determining location of maximumresonant peak (x_(p)=(−b)/2a). The step of determining a value of themaximum resonant peak can include determining the value with of x_(p) aty′_(p).

[0034] Even another embodiment of the invention comprises a computerreadable medium having stored therein instructions for causing aprocessor to execute the methods for determining a location of aresonant peak for a binding partner in a resonant reflectance spectrumwith a colormetric resonant biosensor, as described above.

[0035] Another embodiment of the invention provides a resonantreflection structure comprising a two-dimensional grating arranged in apattern of concentric rings. The difference between an inside diameterand an outside diameter of each concentric ring is equal to aboutone-half of a grating period, wherein each successive ring has an insidediameter that is about one grating period greater than an insidediameter of a previous ring. When the structure is illuminated with anilluminating light beam, a reflected radiation spectrum is produced thatis independent of an illumination polarization angle of the illuminatinglight beam. A resonant grating effect can be produced on the reflectedradiation spectrum, wherein the depth and period of the two-dimensionalgrating are less than the wavelength of the resonant grating effect, andwherein a narrow band of optical wavelengths is reflected from thestructure when the structure is illuminated with a broad band of opticalwavelengths. One or more specific binding substances can be immobilizedon the two-dimensional grating. The two-dimensional grating can have aperiod of about 0.01 microns to about 1 micron and a depth of about 0.1micron to about 1 micron.

[0036] Even another embodiment of the invention provides a transmissionfilter structure comprising a two-dimensional grating arranged in apattern of concentric rings. The difference between an inside diameterand an outside diameter of each concentric ring is equal to aboutone-half of a grating period, wherein each successive ring has an insidediameter that is about one grating period greater than an insidediameter of a previous ring. When the structure is illuminated with anilluminating light beam, a transmitted radiation spectrum is producedthat is independent of an illumination polarization angle of theilluminating light beam. The structure of can be illuminated to producea resonant grating effect on the reflected radiation spectrum, whereinthe depth and period of the two-dimensional grating are less than thewavelength of the resonant grating effect, and wherein a narrow band ofoptical wavelengths is reflected from the structure when the structureis illuminated with a broad band of optical wavelengths. One or morespecific binding substances can be immobilized on the two-dimensionalgrating. The two-dimensional grating can have a period of about 0.01microns to about 1 micron and a depth of about 0.01 microns to about 1micron.

[0037] Still another embodiment of the invention provides a resonantreflection structure comprising an array of holes or posts arranged suchthat the holes or posts are centered on the corners in the center ofhexagons, wherein the hexagons are arranged in a closely packed array.When the structure is illuminated with an illuminating light beam, areflected radiation spectrum is produced that is independent of anillumination polarization angle of the illuminating light beam. Aresonant grating effect can be produced on the reflected radiationspectrum when the structure is illuminated, wherein the depth or heightand period of the array of holes or posts are less than the wavelengthof the resonant grating effect, and wherein a narrow band of opticalwavelengths is reflected from the structure when the structure isilluminated with a broad band of optical wavelengths. The resonantreflection structure can be incorporated into a biosensor wherein one ormore specific binding substances are immobilized on the array of holesor posts. The holes or posts can have a period of about 0.01 microns toabout 1 micron and a depth or height of about 0.01 microns to about 1micron.

[0038] Yet another embodiment of the invention provides a transmissionfilter structure comprising an array of holes or posts arranged suchthat the holes or posts are centered on the corners and in the center ofhexagons, wherein the hexagons are arranged in a closely packed array.When the structure is illuminated with an illuminating light beam, atransmitted radiation spectrum is produced that is independent of anillumination polarization angle of the illuminating light beam. When thestructure is illuminated a resonant grating effect is produced on thereflected radiation spectrum, wherein the depth or height and period ofthe array of holes or posts are less than the wavelength of the resonantgrating effect, and wherein a narrow band of optical wavelengths isreflected from the structure when the structure is illuminated with abroad band of optical wavelengths. The transmission filter structure canbe incorporated into a biosensor, wherein one or more specific bindingsubstances are immobilized on the array of holes or posts. The holes orposts can have a period of about 0.01 microns to about 1 micron and adepth or height of about 0.01 microns to about 1 micron.

[0039] Another embodiment of the invention provides a biosensor oroptical device comprising a first two-dimensional grating comprising ahigh refractive index material and having a top surface and a bottomsurface; and a second two-dimensional grating comprising a highrefractive index material and having a top surface and a bottom surface,wherein the top surface of the second two-dimensional grating isattached to the bottom surface of the first two-dimensional grating.When the biosensor or optical device is illuminated two resonant gratingeffects are produced on the reflected radiation spectrum and the depthand period of both of the two-dimensional gratings are less than thewavelength of the resonant grating effects. A substrate layer cansupport the bottom surface of the second two-dimensional grating. Thebiosensor can further comprise one or more specific binding substancesor one or more specific binding substances bound to their bindingpartners immobilized on the top surface of the first two-dimensionalgrating. The biosensor or optical device can further comprising a coverlayer on the top surface of the first two-dimensional grating, whereinthe one or more specific binding substances are immobilized on thesurface of the cover layer opposite of the two-dimensional grating. Thetop surface of the first two-dimensional grating can be in physicalcontact with a test sample, and the second two dimensional grating maynot be in physical contact with the test sample. A peak resonantreflection wavelength can be measured for the first and secondtwo-dimensional gratings, the difference between the two measurementsindicates the amount of one or more specific binding substances, bindingpartners or both deposited on the surface of the first two-dimensionalgrating.

[0040] Even another embodiment of the invention provides a biosensor oroptical device comprising: a first two-dimensional grating comprising ahigh refractive index material and having a top surface and a bottomsurface, a substrate layer comprising a high refractive index materialand having a top surface and a bottom surface, wherein the top surfaceof the substrate supports the bottom surface of the firsttwo-dimensional grating, and a second two-dimensional grating comprisinga top surface and a bottom surface, wherein the bottom surface of thesecond two-dimensional grating is attached to the bottom surface of thesubstrate. When the biosensor or optical device is illuminated tworesonant grating effects are produced on the reflected radiationspectrum, and wherein the depth and period of both of thetwo-dimensional gratings are less than the wavelength of the resonantgrating effects. The biosensor can comprise one or more specific bindingsubstances or one or more specific binding substances bound to theirbinding partners immobilized on the top surface of the firsttwo-dimensional grating. The biosensor or optical device can furthercomprise a cover layer on the top surface of the first two-dimensionalgrating, wherein the one or more specific binding substances areimmobilized on the surface of the cover layer opposite of thetwo-dimensional grating. The top surface of the first two-dimensionalgrating can be in physical contact with a test sample, and the secondtwo dimensional grating may not be in physical contact with the testsample. When a peak resonant reflection wavelength is measured for thefirst and second two-dimensional gratings, the difference between thetwo measurements can indicate the amount of one or more specific bindingsubstances, binding partners or both deposited on the surface of thefirst two-dimensional grating.

[0041] Still another embodiment of the invention provides a method ofdetecting an interaction of a first molecule with a second testmolecule. The method comprises applying a mixture of the first andsecond molecules to a distinct location on a biosensor, wherein thebiosensor comprises a two-dimensional grating and a substrate layer thatsupports the two-dimensional grating; and wherein, when the biosensor isilluminated a resonant grating effect is produced on the reflectedradiation spectrum, and wherein the depth and period of thetwo-dimensional grating are less than the wavelength of the resonantgrating effect; applying a mixture of the first molecule with a thirdcontrol molecule to a distinct location on the biosensor or a similarbiosensor, wherein the third control molecule does not interact with thefirst molecule, and wherein the third control molecule is about the samesize as the first molecule; and detecting a shift in the reflectedwavelength of light from the distinct locations. Wherein, if the shiftin the reflected wavelength of light from the distinct location to whicha mixture of the first molecule and the second test molecule was appliedis greater than the shift in the reflected wavelength from the distinctlocation to which a mixture of the first molecule with the third controlmolecule was applied, then the first molecule and the second testmolecule interact. The first molecule can be selected from the groupconsisting of a nucleic acid, polypeptide, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus, andbacteria. The second test molecule can be selected from the groupconsisting of a nucleic acid, polypeptide, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus, andbacteria.

[0042] Therefore, unlike surface plasmon resonance, resonant mirrors,and waveguide biosensors, the described compositions and methods enablemany thousands of individual binding reactions to take placesimultaneously upon the biosensor surface. This technology is useful inapplications where large numbers of biomolecular interactions aremeasured in parallel, particularly when molecular labels will alter orinhibit the functionality of the molecules under study. High-throughputscreening of pharmaceutical compound libraries with protein targets, andmicroarray screening of protein-protein interactions for proteomics areexamples of applications that require the sensitivity and throughputafforded by this approach. A biosensor of the invention can bemanufactured, for example, in large areas using a plastic embossingprocess, and thus can be inexpensively incorporated into commondisposable laboratory assay platforms such as microtiter plates andmicroarray slides.

BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIGS. 1A-B show schematic diagrams of one embodiment of anoptical grating structure used for a colormetric resonant reflectancebiosensor. n_(substrate) represents substrate material. n₁ representsthe refractive index of a cover layer. n₂ represents the refractiveindex of a two-dimensional grating. n_(bio) represents the refractiveindex of one or more specific binding substances. t₁ represents thethickness of the cover layer. t₂ represents the thickness of thegrating. t_(bio) represents the thickness of the layer of one or morespecific binding substances. FIG. 1A shows a cross-sectional view of abiosensor. FIG. 1B shows a diagram of a biosensor.

[0044]FIG. 2 shows a schematic drawing of a linear grating structure.

[0045] FIGS. 3A-B shows a grating comprising a rectangular grid ofsquares (FIG. 3A) or holes (FIG. 3B).

[0046]FIG. 4 shows a biosensor cross-section profile utilizing asinusoidally varying grating profile.

[0047]FIG. 5 shows a biosensor cross-section profile in which anembossed substrate is coated with a higher refractive index materialsuch as ZnS or SiN. A cover layer of, for example, epoxy or SOG islayered on top of the higher refractive index material and one or morespecific binding substances are immobilized on the cover layer.

[0048]FIG. 6 shows three types of surface activation chemistry (Amine,Aldehyde, and Nickel) with corresponding chemical linker molecules thatcan be used to covalently attach various types of biomolecule receptorsto a biosensor.

[0049] FIGS. 7A-C shows methods that can be used to amplify the mass ofa binding partner such as detected DNA or detected protein on thesurface of a biosensor.

[0050]FIG. 8 shows a graphic representation of how adsorbed material,such as a protein monolayer, will increase the reflected wavelength ofon a SRVD biosensor.

[0051]FIG. 9 shows an example of a biosensor used as a microarray.

[0052] FIGS. 10A-B shows two biosensor formats that can incorporate acolorimetric resonant reflectance biosensor. FIG. 10A shows a biosensorthat is incorporated into a microtitre plate. FIG. 10B shows a biosensorin a microarray slide format.

[0053]FIG. 11 shows an array of arrays concept for using a biosensorplatform to perform assays with higher density and throughput.

[0054]FIG. 12 shows a diagram of an array of biosensor electrodes. Asingle electrode can comprise a region that contains many gratingperiods and several separate grating regions can occur on the samesubstrate surface.

[0055]FIG. 13 shows a SEM photograph showing the separate gratingregions of an array of biosensor electrodes.

[0056]FIG. 14 shows a biosensor upper surface immersed in a liquidsample. An electrical potential can be applied to the biosensor that iscapable of attracting or repelling a biomolecule near the electrodesurface.

[0057]FIG. 15 shows a biosensor upper surface immersed in a liquidsample. A positive voltage is applied to an electrode and theelectronegative biomolecules are attracted to the biosensor surface.

[0058]FIG. 16 shows a biosensor upper surface immersed in a liquidsample. A negative voltage is applied to an electrode and theelectronegative biomolecules are repelled from the biosensor surfaceusing a negative electrode voltage.

[0059]FIG. 17 demonstrates an example of a biosensor that occurs on thetip of a fiber probe for in vivo detection of biochemical substances.

[0060]FIG. 18 shows an example of the use of two coupled fibers toilluminate and collect reflected light from a biosensor.

[0061]FIG. 19 shows resonance wavelength of a biosensor as a function ofincident angle of detection beam.

[0062]FIG. 20 shows an example of the use of a beam splitter to enableilluminating and reflected light to share a common collimated opticalpath to a biosensor.

[0063]FIG. 21 shows an example of a system for angular scanning of abiosensor.

[0064]FIG. 22 shows SEM photographs of a photoresist grating structurein plan view (center and upper right) and cross-section (lower right).

[0065]FIG. 23 shows a SEM cross-section photograph of a gratingstructure after spin-on glass is applied over a silicon nitride grating.

[0066]FIG. 24 shows examples of biosensor chips (1.5×1.5-inch). Circularareas are regions where the resonant structure is defined.

[0067]FIG. 25 shows response as a function of wavelength of a biosensorthat BSA had been deposited at high concentration, measured in air.Before protein deposition, the resonant wavelength of the biosensor is380 nm and is not observable with the instrument used for thisexperiment.

[0068]FIG. 26 shows response as a function of wavelength comparing anuntreated biosensor with one upon which BSA had been deposited. Bothmeasurements were taken with water on the biosensor's surface.

[0069]FIG. 27 shows response as a function of wavelength of a biosensorthat Borrelia bacteria has been deposited at high concentration andmeasured in water.

[0070]FIG. 28 shows a computer simulation of a biosensor demonstratingthe shift of resonance to longer wavelengths as biomolecules aredeposited on the surface.

[0071]FIG. 29 shows a computer simulation demonstrating the dependenceof peak reflected wavelength on protein coating thickness. Thisparticular biosensor has a dynamic range of 250 nm deposited biomaterialbefore the response begins to saturate.

[0072]FIG. 30 shows an embodiment of a biosensor. n_(substrate)represents the refractive index of a substrate. n₁ represents therefractive index of an optical cover layer. n₂ represents the refractiveindex of a two-dimensional grating. n₃ represents the refractive indexof a high refractive index material such as silicon nitride. n_(bio)represents the refractive index of one or more specific bindingsubstances. t₁ represents the thickness of a cover layer. t₂ representsthe thickness of a two-dimensional grating. t₃ represents the thicknessof a high refractive index material. t_(bio) represents the thickness ofa specific binding substance layer.

[0073]FIG. 31 shows reflected intensity as a function of wavelength fora resonant grating structure when various thicknesses of protein areincorporated onto the upper surface.

[0074]FIG. 32 shows a linear relationship between reflected wavelengthand protein coating thickness for a biosensor shown in FIG. 30.

[0075]FIG. 33 shows instrumentation that can be used to read output of abiosensor. A collimated light source is directed at a biosensor surfaceat normal incidence through an optical fiber, while a second parallelfiber collects the light reflected at normal incidence. A spectrometerrecords the reflectance as a function of wavelength.

[0076]FIG. 34 shows the measured reflectance spectra of a biosensor.

[0077]FIG. 35 shows dependence of peak resonant wavelength measured inliquid upon the concentration of protein BSA dissolved in water.

[0078]FIG. 36 shows dependence of peak resonance wavelength on theconcentration of BSA dissolved in PBS, which was then allowed to dry ona biosensor surface.

[0079] FIGS. 37A-B. FIG. 37A shows a measurement of peak resonantwavelength shift caused by attachment of a streptavidin receptor layerand subsequent detection of a biotinylated IgG. FIG. 37B shows aschematic demonstration of molecules bound to a biosensor.

[0080] FIGS. 38A-B. FIG. 38A shows results of streptavidin detection atvarious concentrations for a biosensor that has been activated with NH₂surface chemistry linked to a biotin receptor molecule. FIG. 38B shows aschematic demonstration of molecules bound to a biosensor.

[0081] FIGS. 39A-B. FIG. 39A shows an assay for detection of anti-goatIgG using a goat antibody receptor molecule. BSA blocking of a detectionsurface yields a clearly measurable background signal due to the mass ofBSA incorporated on the biosensor. A 66 nM concentration of anti-goatIgG is easily measured above the background signal. FIG. 39B shows aschematic demonstration of molecules bound to a biosensor.

[0082] FIGS. 40A-B. FIG. 40A shows a nonlabeled ELISA assay forinterferon-gamma (INF-gamma) using an anti-human IgG INF-gamma receptormolecule, and a neural growth factor (NGF) negative control. FIG. 40Bshows a schematic demonstration of molecules bound to a biosensor.

[0083] FIGS. 41A-B. FIG. 41A shows detection of a 5-amino acid peptide(MW=860) and subsequent cleavage of a pNA label (MW=130) using enzymecaspase-3. FIG. 41B shows a schematic demonstration of molecules boundto a biosensor.

[0084] FIGS. 42A-B. FIG. 42A shows resonant peak in liquid duringcontinuous monitoring of the binding of three separate protein layers.FIG. 42B shows a schematic demonstration of molecules bound to abiosensor.

[0085] FIGS. 43A-B. FIG. 43A shows endpoint resonant frequenciesmathematically determined from the data shown in FIG. 42. FIG. 43B showsa schematic demonstration of molecules bound to a biosensor.

[0086] FIGS. 44A-B. FIG. 44A shows kinetic binding measurement of IgGbinding. FIG. 44B shows a schematic demonstration of molecules bound toa biosensor.

[0087] FIGS. 45A-B. FIG. 45A shows kinetic measurement of a proteasethat cleaves bound protein from a biosensor surface. FIG. 45B shows aschematic demonstration of molecules bound to a biosensor.

[0088]FIG. 46 shows comparison of mathematical fit of parabolic andexponential functions to spectrometer data from a resonant peak. Theexponential curve fit is used to mathematically determine a peakresonant wavelength.

[0089]FIG. 47 shows sensitivity of the mathematically determined peakresonant wavelength to artificially added noise in the measuredspectrum.

[0090]FIG. 48 shows a resonant optical biosensor incorporating anelectrically conducting material.

[0091]FIG. 49 shows a resonant reflection or transmission filterstructure consisting of a set of concentric rings.

[0092]FIG. 50 shows a resonant reflective or transmission filterstructure comprising a hexagonal grid of holes (or a hexagonal grid ofposts) that closely approximates the concentric circle structure of FIG.49 without requiring the illumination beam to be centered upon anyparticular location of the grid.

[0093]FIG. 51 shows a plot of the peak resonant wavelength values fortest solutions. The avidin solution was taken as the baseline referencefor comparison to the Avidin+BSA and Avidin+b-BSA solutions. Addition ofBSA to avidin results in only a small resonant wavelength increase, asthe two proteins are not expected to interact. However, because biotinand avidin bind strongly (Kd=10⁻¹⁵M), the avidin+b-BSA solution willcontain larger bound protein complexes. The peak resonant wavelengthvalue of the avidin+b-BSA solution thus provides a large shift comparedto avidin+BSA.

[0094]FIG. 52 shows a schematic diagram of a detection system.

DETAILED DESCRIPTION OF THE INVENTION

[0095] Subwavelength Structured Surface (SWS) Biosensor

[0096] In one embodiment of the invention, a subwavelength structuredsurface (SWS) is used to create a sharp optical resonant reflection at aparticular wavelength that can be used to track with high sensitivitythe interaction of biological materials, such as specific bindingsubstances or binding partners or both. A colormetric resonantdiffractive grating surface acts as a surface binding platform forspecific binding substances.

[0097] Subwavelength structured surfaces are an unconventional type ofdiffractive optic that can mimic the effect of thin-film coatings. (Peng& Morris, “Resonant scattering from two-dimensional gratings,” J Opt.Soc. Am. A, Vol. 13, No. 5, p. 993, May; Magnusson, & Wang, “Newprinciple for optical filters,” Appl. Phys. Lett., 61, No. 9, p. 1022,August 1992; Peng & Morris, “Experimental demonstration of resonantanomalies in diffraction from two-dimensional gratings,” Optics Letters,Vol. 21, No. 8, p. 549, April, 1996). A SWS structure contains asurface-relief, two-dimensional grating in which the grating period issmall compared to the wavelength of incident light so that nodiffractive orders other than the reflected and transmitted zerothorders are allowed to propagate. A SWS surface narrowband filter cancomprise a two-dimensional grating sandwiched between a substrate layerand a cover layer that fills the grating grooves. Optionally, a coverlayer is not used. When the effective index of refraction of the gratingregion is greater than the substrate or the cover layer, a waveguide iscreated. When a filter is designed properly, incident light passes intothe waveguide region and propagates as a leaky mode. A two-dimensionalgrating structure selectively couples light at a narrow band ofwavelengths into the waveguide. The light propagates only a very shortdistance (on the order of 10-100 micrometers), undergoes scattering, andcouples with the forward- and backward-propagating zeroth-order light.This highly sensitive coupling condition can produce a resonant gratingeffect on the reflected radiation spectrum, resulting in a narrow bandof reflected or transmitted wavelengths. The depth and period of thetwo-dimensional grating are less than the wavelength of the resonantgrating effect.

[0098] The reflected or transmitted color of this structure can bemodulated by the addition of molecules such as specific bindingsubstances or binding partners or both to the upper surface of the coverlayer or the two-dimensional grating surface. The added moleculesincrease the optical path length of incident radiation through thestructure, and thus modify the wavelength at which maximum reflectanceor transmittance will occur.

[0099] In one embodiment, a biosensor, when illuminated with whitelight, is designed to reflect only a single wavelength. When specificbinding substances are attached to the surface of the biosensor, thereflected wavelength (color) is shifted due to the change of the opticalpath of light that is coupled into the grating. By linking specificbinding substances to a biosensor surface, complementary binding partnermolecules can be detected without the use of any kind of fluorescentprobe or particle label. The detection technique is capable of resolvingchanges of, for example, ˜0.1 nm thickness of protein binding, and canbe performed with the biosensor surface either immersed in fluid ordried.

[0100] A detection system consists of, for example, a light source thatilluminates a small spot of a biosensor at normal incidence through, forexample, a fiber optic probe, and a spectrometer that collects thereflected light through, for example, a second fiber optic probe also atnormal incidence. Because no physical contact occurs between theexcitation/detection system and the biosensor surface, no specialcoupling prisms are required and the biosensor can be easily adapted toany commonly used assay platform including, for example, microtiterplates and microarray slides. A single spectrometer reading can beperformed in several milliseconds, thus it is possible to quicklymeasure a large number of molecular interactions taking place inparallel upon a biosensor surface, and to monitor reaction kinetics inreal time.

[0101] This technology is useful in applications where large numbers ofbiomolecular interactions are measured in parallel, particularly whenmolecular labels would alter or inhibit the functionality of themolecules under study. High-throughput screening of pharmaceuticalcompound libraries with protein targets, and microarray screening ofprotein-protein interactions for proteomics are examples of applicationsthat require the sensitivity and throughput afforded by the compositionsand methods of the invention.

[0102] A schematic diagram of an example of a SWS structure is shown inFIG. 1. In FIG. 1, n_(substrate) represents a substrate material. n₁represents the refractive index of an optional cover layer. n₂represents the refractive index of a two-dimensional grating. N_(bio)represents the refractive index of one or more specific bindingsubstances. t₁ represents the thickness of the cover layer above thetwo-dimensional grating structure. t₂ represents the thickness of thegrating. t_(bio) represents the thickness of the layer of one or morespecific binding substances. In one embodiment, are n2<n1 (see FIG. 1).Layer thicknesses (i.e. cover layer, one or more specific bindingsubstances, or a two-dimensional grating) are selected to achieveresonant wavelength sensitivity to additional molecules on the topsurface The grating period is selected to achieve resonance at a desiredwavelength.

[0103] One embodiment of the invention provides a SWS biosensor. A SWSbiosensor comprises a two-dimensional grating, a substrate layer thatsupports the two-dimensional grating, and one or more specific bindingsubstances immobilized on the surface of the two-dimensional gratingopposite of the substrate layer.

[0104] A two-dimensional grating can be comprised of a material,including, for example, zinc sulfide, titanium dioxide, tantalum oxide,and silicon nitride. A cross-sectional profile of a two-dimensionalgrating can comprise any periodically repeating function, for example, a“square-wave.” A two-dimensional grating can be comprised of a repeatingpattern of shapes selected from the group consisting of lines, squares,circles, ellipses, triangles, trapezoids, sinusoidal waves, ovals,rectangles, and hexagons. A sinusoidal cross-sectional profile ispreferable for manufacturing applications that require embossing of agrating shape into a soft material such as plastic. In one embodiment ofthe invention, the depth of the grating is about 0.01 micron to about 1micron and the period of the grating is about 0.01 micron to about 1micron.

[0105] Linear gratings have resonant characteristics where theilluminating light polarization is oriented perpendicular to the gratingperiod. However, a hexagonal grid of holes has better polarizationsymmetry than a rectangular grid of holes. Therefore, a calorimetricresonant reflection biosensor of the invention can comprise, forexample, a hexagonal array of holes (see FIG. 3B) or a grid of parallellines (see FIG. 3A). A linear grating has the same pitch (i.e. distancebetween regions of high and low refractive index), period, layerthicknesses, and material properties as the hexagonal array grating.However, light must be polarized perpendicular to the grating lines inorder to be resonantly coupled into the optical structure. Therefore, apolarizing filter oriented with its polarization axis perpendicular tothe linear grating must be inserted between the illumination source andthe biosensor surface. Because only a small portion of the illuminatinglight source is correctly polarized, a longer integration time isrequired to collect an equivalent amount of resonantly reflected lightcompared to a hexagonal grating.

[0106] While a linear grating can require either a higher intensityillumination source or a longer measurement integration time compared toa hexagonal grating, the fabrication requirements for the linearstructure are simpler. A hexagonal grating pattern is produced byholographic exposure of photoresist to three mutually interfering laserbeams. The three beams are precisely aligned in order to produce agrating pattern that is symmetrical in three directions. A lineargrating pattern requires alignment of only two laser beams to produce aholographic exposure in photoresist, and thus has a reduced alignmentrequirement. A linear grating pattern can also be produced by, forexample, direct writing of photoresist with an electron beam. Also,several commercially available sources exist for producing lineargrating “master” templates for embossing a grating structure intoplastic. A schematic diagram of a linear grating structure is shown inFIG. 2.

[0107] A rectangular grid pattern can be produced in photoresist usingan electron beam direct-write exposure system. A single wafer can beilluminated as a linear grating with two sequential exposures with thepart rotated 90-degrees between exposures.

[0108] A two-dimensional grating can also comprise, for example, a“stepped” profile, in which high refractive index regions of a single,fixed height are embedded within a lower refractive index cover layer.The alternating regions of high and low refractive index provide anoptical waveguide parallel to the top surface of the biosensor. See FIG.5.

[0109] For manufacture, a stepped structure is etched or embossed into asubstrate material such as glass or plastic. See FIG. 5. A uniform thinfilm of higher refractive index material, such as silicon nitride orzinc sulfide is deposited on this structure. The deposited layer willfollow the shape contour of the embossed or etched structure in thesubstrate, so that the deposited material has a surface relief profilethat is identical to the original embossed or etched profile. Thestructure can be completed by the application of an optional cover layercomprised of a material having a lower refractive index than the higherrefractive index material and having a substantially flat upper surface.The covering material can be, for example, glass, epoxy, or plastic.

[0110] This structure allows for low cost biosensor manufacturing,because it can be mass produced. A “master” grating can be produced inglass, plastic, or metal using, for example, a three-beam laserholographic patterning process, See e.g., Cowan, The recording and largescale production of crossed holographic grating arrays using multiplebeam interferometry, Proc. Soc. Photo-optical Instum. Eng. 503:120(1984). A master grating can be repeatedly used to emboss a plasticsubstrate. The embossed substrate is subsequently coated with a highrefractive index material and optionally, a cover layer.

[0111] While a stepped structure is simple to manufacture, it is alsopossible to make a resonant biosensor in which the high refractive indexmaterial is not stepped, but which varies with lateral position. FIG. 4shows a profile in which the high refractive index material of thetwo-dimensional grating, n₂, is sinusoidally varying in height. Toproduce a resonant reflection at a particular wavelength, the period ofthe sinusoid is identical to the period of an equivalent steppedstructure. The resonant operation of the sinusoidally varying structureand its functionality as a biosensor has been verified using GSOLVER(Grating Solver Development Company, Allen, Tex., USA) computer models.

[0112] Techniques for making two-dimensional gratings are disclosed inWang, J. Opt. Soc. Am No. 8, August 1990, pp. 1529-44. Biosensors of theinvention can be made in, for example, a semiconductor microfabricationfacility. Biosensors can also be made on a plastic substrate usingcontinuous embossing and optical coating processes. For this type ofmanufacturing process, a “master” structure is built in a rigid materialsuch as glass or silicon, and is used to generate “mother” structures inan epoxy or plastic using one of several types of replicationprocedures. The “mother” structure, in turn, is coated with a thin filmof conducive material, and used as a mold to electroplate a thick filmof nickel. The nickel “daughter” is released from the plastic “mother”structure. Finally, the nickel “daughter” is bonded to a cylindricaldrum, which is used to continuously emboss the surface relief structureinto a plastic film. A device structure that uses an embossed plasticsubstrate is shown in FIG. 5. Following embossing, the plastic structureis overcoated with a thin film of high refractive index material, andoptionally coated with a planarizing, cover layer polymer, and cut toappropriate size.

[0113] A substrate for a SWS biosensor can comprise, for example, glass,plastic or epoxy. Optionally, a substrate and a two-dimensional gratingcan comprise a single unit. That is, a two dimensional grating andsubstrate are formed from the same material, for example, glass,plastic, or epoxy. The surface of a single unit comprising thetwo-dimensional grating is coated with a material having a highrefractive index, for example, zinc sulfide, titanium dioxide, tantalumoxide, and silicon nitride. One or more specific binding substances canbe immobilized on the surface of the material having a high refractiveindex or on an optional cover layer.

[0114] A biosensor of the invention can further comprise a cover layeron the surface of a two-dimensional grating opposite of a substratelayer. Where a cover layer is present, the one or more specific bindingsubstances are immobilized on the surface of the cover layer opposite ofthe two-dimensional grating. Preferably, a cover layer comprises amaterial that has a lower refractive index than a material thatcomprises the two-dimensional grating. A cover layer can be comprisedof, for example, glass (including spin-on glass (SOG)), epoxy, orplastic.

[0115] For example, various polymers that meet the refractive indexrequirement of a biosensor can be used for a cover layer. SOG can beused due to its favorable refractive index, ease of handling, andreadiness of being activated with specific binding substances using thewealth of glass surface activation techniques. When the flatness of thebiosensor surface is not an issue for a particular system setup, agrating structure of SiN/glass can directly be used as the sensingsurface, the activation of which can be done using the same means as ona glass surface.

[0116] Resonant reflection can also be obtained without a planarizingcover layer over a two-dimensional grating. For example, a biosensor cancontain only a substrate coated with a structured thin film layer ofhigh refractive index material. Without the use of a planarizing coverlayer, the surrounding medium (such as air or water) fills the grating.Therefore, specific binding substances are immobilized to the biosensoron all surfaces a two-dimensional grating exposed to the specificbinding substances, rather than only on an upper surface.

[0117] In general, a biosensor of the invention will be illuminated withwhite light that will contain light of every polarization angle. Theorientation of the polarization angle with respect to repeating featuresin a biosensor grating will determine the resonance wavelength. Forexample, a “linear grating” biosensor structure consisting of a set ofrepeating lines and spaces will have two optical polarizations that cangenerate separate resonant reflections. Light that is polarizedperpendicularly to the lines is called “s-polarized,” while light thatis polarized parallel to the lines is called “p-polarized.” Both the sand p components of incident light exist simultaneously in an unfilteredillumination beam, and each generates a separate resonant signal. Abiosensor structure can generally be designed to optimize the propertiesof only one polarization (the s-polarization), and the non-optimizedpolarization is easily removed by a polarizing filter.

[0118] In order to remove the polarization dependence, so that everypolarization angle generates the same resonant reflection spectra, analternate biosensor structure can be used that consists of a set ofconcentric rings. In this structure, the difference between the insidediameter and the outside diameter of each concentric ring is equal toabout one-half of a grating period. Each successive ring has an insidediameter that is about one grating period greater than the insidediameter of the previous ring. The concentric ring pattern extends tocover a single sensor location—such as a microarray spot or a microtiterplate well. Each separate microarray spot or microtiter plate well has aseparate concentric ring pattern centered within it. e.g., FIG. 49. Allpolarization directions of such a structure have the samecross-sectional profile. The concentric ring structure must beilluminated precisely on-center to preserve polarization independence.The grating period of a concentric ring structure is less than thewavelength of the resonantly reflected light. The grating period isabout 0.01 micron to about 1 micron. The grating depth is about 0.01 toabout 1 micron.

[0119] In another embodiment, an array of holes or posts are arranged toclosely approximate the concentric circle structure described abovewithout requiring the illumination beam to be centered upon anyparticular location of the grid. See e.g. FIG. 50. Such an array patternis automatically generated by the optical interference of three laserbeams incident on a surface from three directions at equal angles. Inthis pattern, the holes (or posts) are centered upon the corners of anarray of closely packed hexagons as shown in FIG. 50. The holes or postsalso occur in the center of each hexagon. Such a hexagonal grid of holesor posts has three polarization directions that “see” the samecross-sectional profile. The hexagonal grid structure, therefore,provides equivalent resonant reflection spectra using light of anypolarization angle. Thus, no polarizing filter is required to removeunwanted reflected signal components. The period of the holes or postscan be about 0.01 microns to about 1 micron and the depth or height canbe about 0.01 microns to about 1 micron.

[0120] The invention provides a resonant reflection structures andtransmission filter structures comprising concentric circle gratings andhexagonal grids of holes or posts. For a resonant reflection structure,light output is measured on the same side of the structure as theilluminating light beam. For a transmission filter structure, lightoutput is measured on the opposite side of the structure as theilluminating beam. The reflected and transmitted signals arecomplementary. That is, if a wavelength is strongly reflected, it isweakly transmitted. Assuming no energy is absorbed in the structureitself, the reflected+transmitted energy at any given wavelength isconstant. The resonant reflection structure and transmission filters aredesigned to give a highly efficient reflection at a specifiedwavelength. Thus, a reflection filter will “pass” a narrow band ofwavelengths, while a transmission filter will “cut” a narrow band ofwavelengths from incident light.

[0121] A resonant reflection structure or a transmission filterstructure can comprise a two-dimensional grating arranged in a patternof concentric circles. A resonant reflection structure or transmissionfilter structure can also comprise a hexagonal grid of holes or posts.When these structure are illuminated with an illuminating light beam, areflected radiation spectrum is produced that is independent of anillumination polarization angle of the illuminating light beam. Whenthese structures are illuminated a resonant grating effect is producedon the reflected radiation spectrum, wherein the depth and period of thetwo-dimensional grating or hexagonal grid of holes or posts are lessthan the wavelength of the resonant grating effect. These structuresreflect a narrow band of light when the structure is illuminated with abroadband of light.

[0122] Resonant reflection structures and transmission filter structuresof the invention can be used as biosensors. For example, one or morespecific binding substances can be immobilized on the hexagonal grid ofholes or posts or on the two-dimensional grating arranged in concentriccircles.

[0123] In one embodiment of the invention, a reference resonant signalis provided for more accurate measurement of peak resonant wavelengthshifts. The reference resonant signal can cancel out environmentaleffects, including, for example, temperature. A reference signal can beprovided using a resonant reflection superstructure that produces twoseparate resonant wavelengths. A transparent resonant reflectionsuperstructure can contain two sub-structures. A first sub-structurecomprises a first two-dimensional grating with a top and a bottomsurface. The top surface of a two-dimensional grating comprises thegrating surface. The first two-dimensional grating can comprise one ormore specific binding substances immobilized on its top surface. The topsurface of the first two-dimensional grating is in contact with a testsample. An optional substrate layer can be present to support the bottomsurface of the first two-dimensional grating. The substrate layercomprises a top and bottom surface. The top surface of the substrate isin contact with, and supports the bottom surface of the firsttwo-dimensional grating.

[0124] A second sub-structure comprises a second two-dimensional gratingwith a top surface and a bottom surface. The second two-dimensionalgrating is not in contact with a test sample. The second two-dimensionalgrating can be fabricated onto the bottom surface of the substrate thatsupports the first two-dimensional grating. Where the secondtwo-dimensional grating is fabricated on the substrate that supports thefirst two-dimensional grating, the bottom surface of the secondtwo-dimensional grating can be fabricated onto the bottom surface of thesubstrate. Therefore, the top surface of the second two-dimensionalgrating will face the opposite direction of the top surface of the firsttwo-dimensional grating.

[0125] The top surface of the second two-dimensional grating can also beattached directly to the bottom surface of the first sub-structure. Inthis embodiment the top surface of the second two-dimensional gratingwill face the same direction as the top surface of the firsttwo-dimensional grating. A substrate can support the bottom surface ofthe second two-dimensional grating in this embodiment.

[0126] Because the second sub-structure is not in physical contact withthe test sample, its peak resonant wavelength is not subject to changesin the optical density of the test media, or deposition of specificbinding substances or binding partners on the surface of the firsttwo-dimensional grating. Therefore, such a superstructure produces tworesonant signals. Because the location of the peak resonant wavelengthin the second sub-structure is fixed, the difference in peak resonantwavelength between the two sub-structures provides a relative means fordetermining the amount of specific binding substances or bindingpartners or both deposited on the top surface of the first substructurethat is exposed to the test sample.

[0127] A biosensor superstructure can be illuminated from its topsurface or from its bottom surface, or from both surfaces. The peakresonance reflection wavelength of the first substructure is dependenton the optical density of material in contact with the superstructuresurface, while the peak resonance reflection wavelength of the secondsubstructure is independent of the optical density of material incontact with the superstructure surface.

[0128] In one embodiment of the invention, a biosensor is illuminatedfrom the bottom surface of the biosensor. Approximately 50% of theincident light is reflected from the bottom surface of biosensor withoutreaching the active (top) surface of the biosensor. A thin film orphysical structure can be included in a biosensor composition that iscapable of maximizing the amount of light that is transmitted to theupper surface of the biosensor while minimizing the reflected energy atthe resonant wavelength. The anti-reflection thin film or physicalstructure of the bottom surface of the biosensor can comprise, forexample, a single dielectric thin film, a stack of multiple dielectricthin films, or a “motheye” structure that is embossed into the bottombiosensor surface. An example of a motheye structure is disclosed inHobbs, et al. “Automated interference lithography system for generationof sub-micron feature size patterns,” Proc. 1999Micromachine Technologyfor Diffracting and Holographic Optics, Society of Photo-OpticalInstrumentation Engineers, p. 124-135, (1999).

[0129] In one embodiment of the invention, an optical device isprovided. An optical device comprises a structure similar to anybiosensor of the invention; however, an optical device does not compriseone of more binding substances immobilized on the two-dimensionalgrating. An optical device can be used as a narrow band optical filter.

[0130] In one embodiment of the invention, an interaction of a firstmolecule with a second test molecule can be detected. A SWS biosensor asdescribed above is used; however, there are no specific bindingsubstances immobilized on its surface. Therefore, the biosensorcomprises a two-dimensional grating, a substrate layer that supports thetwo-dimensional grating, and optionally, a cover layer. As describedabove, when the biosensor is illuminated a resonant grating effect isproduced on the reflected radiation spectrum, and the depth and periodof the two-dimensional grating are less than the wavelength of theresonant grating effect.

[0131] To detect an interaction of a first molecule with a second testmolecule, a mixture of the first and second molecules is applied to adistinct location on a biosensor. A distinct location can be one spot orwell on a biosensor or can be a large area on a biosensor. A mixture ofthe first molecule with a third control molecule is also applied to adistinct location on a biosensor. The biosensor can be the samebiosensor as described above, or can be a second biosensor. If thebiosensor is the same biosensor, a second distinct location can be usedfor the mixture of the first molecule and the third control molecule.Alternatively, the same distinct biosensor location can be used afterthe first and second molecules are washed from the biosensor. The thirdcontrol molecule does not interact with the first molecule and is aboutthe same size as the first molecule. A shift in the reflected wavelengthof light from the distinct locations of the biosensor or biosensors ismeasured. If the shift in the reflected wavelength of light from thedistinct location having the first molecule and the second test moleculeis greater than the shift in the reflected wavelength from the distinctlocation having the first molecule and the third control molecule, thenthe first molecule and the second test molecule interact. Interactioncan be, for example, hybridization of nucleic acid molecules, specificbinding of an antibody or antibody fragment to an antigen, and bindingof polypeptides. A first molecule, second test molecule, or thirdcontrol molecule can be, for example, a nucleic acid, polypeptide,antigen, polyclonal antibody, monoclonal antibody, single chain antibody(scFv), F(ab) fragment, F(ab′)₂ fragment, Fv fragment, small organicmolecule, cell, virus, and bacteria.

[0132] Specific Binding Substances and Binding Partners

[0133] One or more specific binding substances are immobilized on thetwo-dimensional grating or cover layer, if present, by for example,physical adsorption or by chemical binding. A specific binding substancecan be, for example, a nucleic acid, polypeptide, antigen, polyclonalantibody, monoclonal antibody, single chain antibody (scFv), F(ab)fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule, cell,virus, bacteria, or biological sample. A biological sample can be forexample, blood, plasma, serum, gastrointestinal secretions, homogenatesof tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid,amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavagefluid, semen, lymphatic fluid, tears, or prostatitc fluid.

[0134] Preferably, one or more specific binding substances are arrangedin a microarray of distinct locations on a biosensor. A microarray ofspecific binding substances comprises one or more specific bindingsubstances on a surface of a biosensor of the invention such that asurface contains many distinct locations, each with a different specificbinding substance or with a different amount of a specific bindingsubstance. For example, an array can comprise 1, 10, 100, 1,000, 10,000,or 100,000 distinct locations. Such a biosensor surface is called amicroarray because one or more specific binding substances are typicallylaid out in a regular grid pattern in x-y coordinates. However, amicroarray of the invention can comprise one or more specific bindingsubstance laid out in any type of regular or irregular pattern. Forexample, distinct locations can define a microarray of spots of one ormore specific binding substances. A microarray spot can be about 50 toabout 500 microns in diameter. A microarray spot can also be about 150to about 200 microns in diameter. One or more specific bindingsubstances can be bound to their specific binding partners.

[0135] A microarray on a biosensor of the invention can be created byplacing microdroplets of one or more specific binding substances onto,for example, an x-y grid of locations on a two-dimensional grating orcover layer surface. When the biosensor is exposed to a test samplecomprising one or more binding partners, the binding partners will bepreferentially attracted to distinct locations on the microarray thatcomprise specific binding substances that have high affinity for thebinding partners. Some of the distinct locations will gather bindingpartners onto their surface, while other locations will not.

[0136] A specific binding substance specifically binds to a bindingpartner that is added to the surface of a biosensor of the invention. Aspecific binding substance specifically binds to its binding partner,but does not substantially bind other binding partners added to thesurface of a biosensor. For example, where the specific bindingsubstance is an antibody and its binding partner is a particularantigen, the antibody specifically binds to the particular antigen, butdoes not substantially bind other antigens. A binding partner can be,for example, a nucleic acid, polypeptide, antigen, polyclonal antibody,monoclonal antibody, single chain antibody (scFv), F(ab) fragment,F(ab′)₂ fragment, Fv fragment, small organic molecule, cell, virus,bacteria, and biological sample. A biological sample can be, forexample, blood, plasma, serum, gastrointestinal secretions, homogenatesof tissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid,amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavagefluid, semen, lymphatic fluid, tears, and prostatitc fluid.

[0137] One example of a microarray of the invention is a nucleic acidmicroarray, in which each distinct location within the array contains adifferent nucleic acid molecule. In this embodiment, the spots withinthe nucleic acid microarray detect complementary chemical binding withan opposing strand of a nucleic acid in a test sample.

[0138] While microtiter plates are the most common format used forbiochemical assays, microarrays are increasingly seen as a means formaximizing the number of biochemical interactions that can be measuredat one time while minimizing the volume of precious reagents. Byapplication of specific binding substances with a microarray spotteronto a biosensor of the invention, specific binding substance densitiesof 10,000 specific binding substances/in² can be obtained. By focusingan illumination beam to interrogate a single microarray location, abiosensor can be used as a label-free microarray readout system.

[0139] Immobilization or One or More Specific Binding Substances

[0140] Immobilization of one or more binding substances onto a biosensoris performed so that a specific binding substance will not be washedaway by rinsing procedures, and so that its binding to binding partnersin a test sample is unimpeded by the biosensor surface. Severaldifferent types of surface chemistry strategies have been implementedfor covalent attachment of specific binding substances to, for example,glass for use in various types of microarrays and biosensors. These samemethods can be readily adapted to a biosensor of the invention. Surfacepreparation of a biosensor so that it contains the correct functionalgroups for binding one or more specific binding substances is anintegral part of the biosensor manufacturing process.

[0141] One or more specific binding substances can be attached to abiosensor surface by physical adsorption (i.e., without the use ofchemical linkers) or by chemical binding (i.e., with the use of chemicallinkers). Chemical binding can generate stronger attachment of specificbinding substances on a biosensor surface and provide definedorientation and conformation of the surface-bound molecules.

[0142] Several examples of chemical binding of specific bindingsubstances to a biosensor of the invention appear in Example 8, below.Other types of chemical binding include, for example, amine activation,aldehyde activation, and nickel activation. These surfaces can be usedto attach several different types of chemical linkers to a biosensorsurface, as shown in FIG. 6. While an amine surface can be used toattach several types of linker molecules, an aldehyde surface can beused to bind proteins directly, without an additional linker. A nickelsurface can be used to bind molecules that have an incorporatedhistidine (“his”) tag. Detection of “his-tagged” molecules with anickel-activated surface is well known in the art (Whitesides, Anal.Chem. 68, 490, (1996)).

[0143] Imobilization of specific binding substances to plastic, epoxy,or high refractive index material can be performed essentially asdescribed for immobilization to glass. However, the acid wash step canbe eliminated where such a treatment would damage the material to whichthe specific binding substances are immobilized.

[0144] For the detection of binding partners at concentrations less thanabout ˜0.1 ng/ml, it is preferable to amplify and transduce bindingpartners bound to a biosensor into an additional layer on the biosensorsurface. The increased mass deposited on the biosensor can be easilydetected as a consequence of increased optical path length. Byincorporating greater mass onto a biosensor surface, the optical densityof binding partners on the surface is also increased, thus rendering agreater resonant wavelength shift than would occur without the addedmass. The addition of mass can be accomplished, for example,enzymatically, through a “sandwich” assay, or by direct application ofmass to the biosensor surface in the form of appropriately conjugatedbeads or polymers of various size and composition. This principle hasbeen exploited for other types of optical biosensors to demonstratesensitivity increases over 1500× beyond sensitivity limits achievedwithout mass amplification. See, e.g., Jenison et al.,“Interference-based detection of nucleic acid targets on opticallycoated silicon,” Nature Biotechnology, 19: 62-65, 2001.

[0145] As an example, FIG. 7A shows that an NH₂-activated biosensorsurface can have a specific binding substance comprising a single-strandDNA capture probe immobilized on the surface. The capture probeinteracts selectively with its complementary target binding partner. Thebinding partner, in turn, can be designed to include a sequence or tagthat will bind a “detector” molecule. As shown in FIG. 7A, a detectormolecule can contain, for example, a linker to horseradish peroxidase(HRP) that, when exposed to the correct enzyme, will selectively depositadditional material on the biosensor only where the detector molecule ispresent. Such a procedure can add, for example, 300 angstroms ofdetectable biomaterial to the biosensor within a few minutes.

[0146] A “sandwich” approach can also be used to enhance detectionsensitivity. In this approach, a large molecular weight molecule can beused to amplify the presence of a low molecular weight molecule. Forexample, a binding partner with a molecular weight of, for example,about 0.1 kDa to about 20 kDa, can be tagged with, for example,succinimidyl-6-[a-methyl-a-(2-pyridyl-dithio) toluamido] hexanoate(SMPT), or dimethylpimelimidate (DMP), histidine, or a biotin molecule,as shown in FIG. 7B. Where the tag is biotin, the biotin molecule willbinds strongly with streptavidin, which has a molecular weight of 60kDa. Because the biotin/streptavidin interaction is highly specific, thestreptavidin amplifies the signal that would be produced only by thesmall binding partner by a factor of 60.

[0147] Detection sensitivity can be further enhanced through the use ofchemically derivatized small particles. “Nanoparticles” made ofcolloidal gold, various plastics, or glass with diameters of about 3-300nm can be coated with molecular species that will enable them tocovalently bind selectively to a binding partner. For example, as shownin FIG. 7C, nanoparticles that are covalently coated with streptavidincan be used to enhance the visibility of biotin-tagged binding partnerson the biosensor surface. While a streptavidin molecule itself has amolecular weight of 60 kDa, the derivatized bead can have a molecularweight of any size, including, for example, 60 KDa. Binding of a largebead will result in a large change in the optical density upon thebiosensor surface, and an easily measurable signal. This method canresult in an approximately 1000× enhancement in sensitivity resolution.

[0148] Surface-Relief Volume Diffractive Biosensors

[0149] Another embodiment of the invention is a biosensor that comprisesvolume surface-relief volume diffractive structures (a SRVD biosensor).SRVD biosensors have a surface that reflect predominantly at aparticular narrow band of optical wavelengths when illuminated with abroad band of optical wavelengths. Where specific binding substancesand/or binding partners are immobilized on a SRVD biosensor, thereflected wavelength of light is shifted. One-dimensional surfaces, suchas thin film interference filters and Bragg reflectors, can select anarrow range of reflected or transmitted wavelengths from a broadbandexcitation source, however, the deposition of additional material, suchas specific binding substances and/or binding partners onto their uppersurface results only in a change in the resonance linewidth, rather thanthe resonance wavelength. In contrast, SRVD biosensors have the abilityto alter the reflected wavelength with the addition of material, such asspecific binding substances and/or binding partners to the surface.

[0150] A SRVD biosensor comprises a sheet material having a first andsecond surface. The first surface of the sheet material defines reliefvolume diffraction structures. A sheet material can be comprised of, forexample, plastic, glass, semiconductor wafer, or metal film.

[0151] A relief volume diffractive structure can be, for example, atwo-dimensional grating, as described above, or a three-dimensionalsurface-relief volume diffractive grating. The depth and period ofrelief volume diffraction structures are less than the resonancewavelength of light reflected from a biosensor.

[0152] A three-dimensional surface-relief volume diffractive grating canbe, for example, a three-dimensional phase-quantized terraced surfacerelief pattern whose groove pattern resembles a stepped pyramid. Whensuch a grating is illuminated by a beam of broadband radiation, lightwill be coherently reflected from the equally spaced terraces at awavelength given by twice the step spacing times the index of refractionof the surrounding medium. Light of a given wavelength is resonantlydiffracted or reflected from the steps that are a half-wavelength apart,and with a bandwidth that is inversely proportional to the number ofsteps. The reflected or diffracted color can be controlled by thedeposition of a dielectric layer so that a new wavelength is selected,depending on the index of refraction of the coating.

[0153] A stepped-phase structure can be produced first in photoresist bycoherently exposing a thin photoresist film to three laser beams, asdescribed previously. See e.g., Cowen, “The recording and large scalereplication of crossed holographic grating arrays using multiple beaminterferometry,” in International Conference on the Application, Theory,and Fabrication of Periodic Structures, Diffraction Gratings, and MoirePhenomena II, Lerner, ed., Proc. Soc. Photo-Opt. Instrum. Eng., 503,120-129, 1984; Cowen, “Holographic honeycomb microlens,” Opt. Eng. 24,796-802 (1985); Cowen & Slafer, “The recording and replication ofholographic micropatterns for the ordering of photographic emulsiongrains in film systems,” J Imaging Sci. 31, 100-107, 1987. The nonlinearetching characteristics of photoresist are used to develop the exposedfilm to create a three-dimensional relief pattern. The photoresiststructure is then replicated using standard embossing procedures. Forexample, a thin silver film is deposited over the photoresist structureto form a conducting layer upon which a thick film of nickel can beelectroplated. The nickel “master” plate is then used to emboss directlyinto a plastic film, such as vinyl, that has been softened by heating orsolvent.

[0154] The theory describing the design and fabrication ofthree-dimensional phase-quantized terraced surface relief pattern thatresemble stepped pyramids is described: Cowen, “Aztec surface-reliefvolume diffractive structure,” J Opt. Soc. Am. A, 7:1529 (1990).

[0155] An example of a three-dimensional phase-quantized terracedsurface relief pattern is a pattern that resembles a stepped pyramid.Each inverted pyramid is approximately 1 micron in diameter, preferably,each inverted pyramid can be about 0.5 to about 5 microns diameter,including for example, about 1 micron. The pyramid structures can beclose-packed so that a typical microarray spot with a diameter of150-200 microns can incorporate several hundred stepped pyramidstructures. The relief volume diffraction structures have a period ofabout 0.1 to about 1 micron and a depth of about 0.1 to about 1 micron.FIG. 8 demonstrates how individual microarray locations (with an entiremicroarray spot incorporating hundreds of pyramids now represented by asingle pyramid for one microarray spot) can be optically queried todetermine if specific binding substances or binding partners areadsorbed onto the surface. When the structure is illuminated with whitelight, structures without significant bound material will reflectwavelengths determined by the step height of the structure. When higherrefractive index material, such as binding partners or specific bindingsubstances, are incorporated over the reflective metal surface, thereflected wavelength is modified to shift toward longer wavelengths. Thecolor that is reflected from the terraced step structure istheoretically given as twice the step height times the index ofrefraction of a reflective material that is coated onto the firstsurface of a sheet material of a SRVD biosensor. A reflective materialcan be, for example silver, aluminum, or gold.

[0156] One or more specific binding substances, as described above, areimmobilized on the reflective material of a SRVD biosensor. One or morespecific binding substances can be arranged in microarray of distinctlocations, as described above, on the reflective material. FIG. 9provides an example of a 9-element microarray biosensor. Many individualgrating structures, represented by small circles, lie within eachmicroarray spot. The microarray spots, represented by the largercircles, will reflect white light in air at a wavelength that isdetermined by the refractive index of material on their surface.Microarray locations with additional adsorbed material will havereflected wavelengths that are shifted toward longer wavelengths,represented by the larger circles.

[0157] Because the reflected wavelength of light from a SRVD biosensoris confined to a narrow bandwidth, very small changes in the opticalcharacteristics of the surface manifest themselves in easily observedchanges in reflected wavelength spectra. The narrow reflection bandwidthprovides a surface adsorption sensitivity advantage compared toreflectance spectrometry on a flat surface.

[0158] A SRVD biosensor reflects light predominantly at a first singleoptical wavelength when illuminated with a broad band of opticalwavelengths, and reflects light at a second single optical wavelengthwhen one or more specific binding substances are immobilized on thereflective surface. The reflection at the second optical wavelengthresults from optical interference. A SRVD biosensor also reflects lightat a third single optical wavelength when the one or more specificbinding substances are bound to their respective binding partners, dueto optical interference.

[0159] Readout of the reflected color can be performed serially byfocusing a microscope objective onto individual microarray spots andreading the reflected spectrum, or in parallel by, for example,projecting the reflected image of the microarray onto a high resolutioncolor CCD camera.

[0160] A SRVD biosensor can be manufactured by, for example, producing ametal master plate, and stamping a relief volume diffractive structureinto, for example, a plastic material like vinyl. After stamping, thesurface is made reflective by blanket deposition of, for example, a thinmetal film such as gold, silver, or aluminum. Compared to MEMS-basedbiosensors that rely upon photolithography, etching, and wafer bondingprocedures, the manufacture of a SRVD biosensor is very inexpensive.

[0161] Liquid-Containing Vessels

[0162] A SWS or SRVD biosensor of the invention can comprise an innersurface, for example, a bottom surface of a liquid-containing vessel. Aliquid-containing vessel can be, for example, a microtiter plate well, atest tube, a petri dish, or a microfluidic channel. One embodiment ofthis invention is a SWS or SRVD biosensor that is incorporated into anytype of microtiter plate. For example, a SWS biosensor or SRVD biosensorcan be incorporated into the bottom surface of a microtiter plate byassembling the walls of the reaction vessels over the resonantreflection surface, as shown in FIG. 10, so that each reaction “spot”can be exposed to a distinct test sample. Therefore, each individualmicrotiter plate well can act as a separate reaction vessel. Separatechemical reactions can, therefore, occur within adjacent wells withoutintermixing reaction fluids and chemically distinct test solutions canbe applied to individual wells.

[0163] Several methods for attaching a biosensor of the invention to thebottom surface of bottomless microtiter plates can be used, including,for example, adhesive attachment, ultrasonic welding, and laser welding.

[0164] The most common assay formats for pharmaceutical high-throughputscreening laboratories, molecular biology research laboratories, anddiagnostic assay laboratories are microtiter plates. The plates arestandard-sized plastic cartridges that can contain 96, 384, or 1536individual reaction vessels arranged in a grid. Due to the standardmechanical configuration of these plates, liquid dispensing, roboticplate handling, and detection systems are designed to work with thiscommon format. A biosensor of the invention can be incorporated into thebottom surface of a standard microtiter plate. See, e g., FIG. 10.Because the biosensor surface can be fabricated in large areas, andbecause the readout system does not make physical contact with thebiosensor surface, an arbitrary number of individual biosensor areas canbe defined that are only limited by the focus resolution of theillumination optics and the x-y stage that scans theillumination/detection probe across the biosensor surface.

[0165] Holding Fixtures

[0166] Any number of biosensors that are, for example, about 1 mm² toabout 5 mm², and preferably less than about 3×mm² can be arranged onto aholding fixture that can simultaneously dip the biosensors into separateliquid-containing vessels, such as wells of a microtiter plate, forexample, a 96- , 384-, or 1536-well microtiter plate. See e.g., FIG. 11.Each of the biosensors can contain multiple distinct locations. Aholding fixture has one or more biosensors attached to the holdingfixture so that each individual biosensor can be lowered into a separateliquid-containing vessel. A holding fixture can comprise plastic, epoxyor metal. For example, 50, 96, 384, or 1,000, or 1,536 biosensors can bearranged on a holding fixture, where each biosensor has 25, 100, 500, or1,000 distinct locations. As an example, where 96 biosenors are attachedto a holding fixture and each biosensor comprises 100 distinctlocations, 9600 biochemical assays can be performed simultaneously.

[0167] Methods of using SWS and SRVD Biosensors

[0168] SWS and SRVD biosensors of the invention can be used to study oneor a number of specific binding substance/binding partner interactionsin parallel. Binding of one or more specific binding substances to theirrespective binding partners can be detected, without the use of labels,by applying one or more binding partners to a SWS or SRVD biosensor thathave one or more specific binding substances immobilized on theirsurfaces. A SWS biosensor is illuminated with light and a maxima inreflected wavelength, or a minima in transmitted wavelength of light isdetected from the biosensor. If one or more specific binding substanceshave bound to their respective binding partners, then the reflectedwavelength of light is shifted as compared to a situation where one ormore specific binding substances have not bound to their respectivebinding partners. Where a SWS biosensor is coated with an array ofdistinct locations containing the one or more specific bindingsubstances, then a maxima in reflected wavelength or minima intransmitted wavelength of light is detected from each distinct locationof the biosensor.

[0169] A SRVD biosensor is illuminated with light after binding partnershave been added and the reflected wavelength of light is detected fromthe biosensor. Where one or more specific binding substances have boundto their respective binding partners, the reflected wavelength of lightis shifted.

[0170] In one embodiment of the invention, a variety of specific bindingsubstances, for example, antibodies, can be immobilized in an arrayformat onto a biosensor of the invention. The biosensor is thencontacted with a test sample of interest comprising binding partners,such as proteins. Only the proteins that specifically bind to theantibodies immobilized on the biosensor remain bound to the biosensor.Such an approach is essentially a large-scale version of anenzyme-linked immunosorbent assay; however, the use of an enzyme orfluorescent label is not required.

[0171] The Activity of an enzyme can be detected by applying one or moreenzymes to a SWS or SRVD biosensor to which one or more specific bindingsubstances have been immobilized. The biosensor is washed andilluminated with light. The reflected wavelength of light is detectedfrom the biosensor. Where the one or more enzymes have altered the oneor more specific binding substances of the biosensor by enzymaticactivity, the reflected wavelength of light is shifted.

[0172] Additionally, a test sample, for example, cell lysates containingbinding partners can be applied to a biosensor of the invention,followed by washing to remove unbound material. The binding partnersthat bind to a biosensor can be eluted from the biosensor and identifiedby, for example, mass spectrometry. Optionally, a phage DNA displaylibrary can be applied to a biosensor of the invention followed bywashing to remove unbound material. Individual phage particles bound tothe biosensor can be isolated and the inserts in these phage particlescan then be sequenced to determine the identity of the binding partner.

[0173] For the above applications, and in particular proteomicsapplications, the ability to selectively bind material, such as bindingpartners from a test sample onto a biosensor of the invention, followedby the ability to selectively remove bound material from a distinctlocation of the biosensor for further analysis is advantageous.Biosensors of the invention are also capable of detecting andquantifying the amount of a binding partner from a sample that is boundto a biosensor array distinct location by measuring the shift inreflected wavelength of light. For example, the wavelength shift at onedistinct biosensor location can be compared to positive and negativecontrols at other distinct biosensor locations to determine the amountof a binding partner that is bound to a biosensor array distinctlocation.

[0174] SWS and Electrically Conducting Material

[0175] An optional biosensor structure can further enable a biosensorarray to selectively attract or repel binding partners from individualdistinct locations on a biosensor. As is well known in the art, anelectromotive force can be applied to biological molecules such asnucleic acids and amino acids subjecting them to an electric field.Because these molecules are electronegative, they are attracted to apositively charged electrode and repelled by a negatively chargedelectrode.

[0176] A grating structure of a resonant optical biosensor can be builtusing an electrically conducting material rather than an electricallyinsulating material. An electric field can be applied near the biosensorsurface. Where a grating operates as both a resonant reflector biosensorand as an electrode, the grating comprises a material that is bothoptically transparent near the resonant wavelength, and has lowresistivity. In one embodiment of the invention, the material is indiumtin oxide, InSn_(x)O_(1-x) (ITO). ITO is commonly used to producetransparent electrodes for flat panel optical displays, and is thereforereadily available at low cost on large glass sheets. The refractiveindex of ITO can be adjusted by controlling x, the fraction of Sn thatis present in the material. Because the liquid test sample solution willhave mobile ions (and will therefore be an electrical conductor) it isnecessary for the ITO electrodes to be coated with an insulatingmaterial. For the resonant optical biosensor, a grating layer is coatedwith a layer with lower refractive index material. Materials such ascured photoresist (n=1.65), cured optical epoxy (n=1.5), and glass(n=1.4-1.5) are strong electrical insulators that also have a refractiveindex that is lower than ITO (n=2.0-2.65). A cross-sectional diagram ofa biosensor that incorporates an ITO grating is shown in FIG. 48. n₁represents the refractive index of an electrical insulator. n₂represents the refractive index of a two-dimensional grating. t₁represents the thickness of the electrical insulator. t₂ represents thethickness of the two-dimensional grating. n_(bio) represents therefractive index of one or more specific binding substances and t_(BIO)represents the thickness of the one or more specific binding substances.

[0177] A grating can be a continuous sheet of ITO that contains an arrayof regularly spaced holes. The holes are filled in with an electricallyinsulating material, such as cured photoresist. The electricallyinsulating layer overcoats the ITO grating so that the upper surface ofthe structure is completely covered with electrical insulator, and sothat the upper surface is substantially flat. When the biosensor isilluminated with light a resonant grating effect is produced on thereflected radiation spectrum. The depth and the period of the gratingare less than the wavelength of the resonant grating effect.

[0178] As shown in FIG. 12 and FIG. 13, a single electrode can comprisea region that contains many grating periods. Building two or moreseparate grating regions on the same substrate surface creates an arrayof biosensor electrodes. Electrical contact to each biosensor electrodeis provided using an electrically conducting trace that is built fromthe same material as the conductor within the biosensor electrode. Theconducting trace is connected to a voltage source that can apply anelectrical potential to the electrode. To apply an electrical potentialto the biosensor that is capable of attracting or repelling a moleculenear the electrode surface, a biosensor upper surface can be immersed ina liquid sample as shown in FIG. 14. A “common” electrode can be placedwithin the sample liquid, and a voltage can be applied between oneselected biosensor electrode region and the common electrode. In thisway, one, several, or all electrodes can be activated or inactivated ata given time. FIG. 15 illustrates the attraction of electronegativemolecules to the biosensor surface when a positive voltage is applied tothe electrode, while FIG. 16 illustrates the application of a repellingforce such as a reversed electrical charge to electronegative moleculesusing a negative electrode voltage.

[0179] Detection Systems

[0180] A detection system can comprise a biosensor of the invention, alight source that directs light to the biosensor, and a detector thatdetects light reflected from the biosensor. In one embodiment, it ispossible to simplify the readout instrumentation by the application of afilter so that only positive results over a determined threshold triggera detection.

[0181] A light source can illuminate a biosensor from its top surface,i.e., the surface to which one or more specific binding substances areimmobilized or from its bottom surface. By measuring the shift inresonant wavelength at each distinct location of a biosensor of theinvention, it is possible to determine which distinct locations havebinding partners bound to them. The extent of the shift can be used todetermine the amount of binding partners in a test sample and thechemical affinity between one or more specific binding substances andthe binding partners of the test sample.

[0182] A biosensor of the invention can be illuminated twice. The firstmeasurement determines the reflectance spectra of one or more distinctlocations of a biosensor array with one or more specific bindingsubstances immobilized on the biosensor. The second measurementdetermines the reflectance spectra after one or more binding partnersare applied to a biosensor. The difference in peak wavelength betweenthese two measurements is a measurement of the amount of bindingpartners that have specifically bound to a biosensor or one or moredistinct locations of a biosensor. This method of illumination cancontrol for small nonuniformities in a surface of a biosensor that canresult in regions with slight variations in the peak resonantwavelength. This method can also control for varying concentrations ormolecular weights of specific binding substances immobilized on abiosensor

[0183] Computer simulation can be used to determine the expecteddependence between a peak resonance wavelength and an angle of incidentillumination. A biosensor structure as shown in FIG. 1 can be forpurposes of demonstration. The substrate chosen was glass(n_(substrate)=1.50). The grating is a two-dimensional pattern ofsilicon nitride squares (t₂=180 nm, n₂=2.01 (n=refractive index),k₂=0.001 (k=absorption coefficient)) with a period of 510 nm, and afilling factor of 56.2% (i.e., 56.2% of the surface is covered withsilicon nitride squares while the rest is the area between the squares).The areas between silicon nitride squares are filled with a lowerrefractive index material. The same material also covers the squares andprovides a uniformly flat upper surface. For this simulation, a glasslayer was selected (n₁=1.40) that covers the silicon nitride squares byt₂=100 nm.

[0184] The reflected intensity as a function of wavelength was modeledusing GSOLVER software, which utilizes full 3-dimensional vector codeusing hybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination can be from any incidence and any polarization.

[0185]FIG. 19 plots the dependence of the peak resonant wavelength uponthe incident illumination angle. The simulation shows that there is astrong correlation between the angle of incident light, and the peakwavelength that is measured. This result implies that the collimation ofthe illuminating beam, and the alignment between the illuminating beamand the reflected beam will directly affect the resonant peak linewidththat is measured. If the collimation of the illuminating beam is poor, arange illuminating angles will be incident on the biosensor surface, anda wider resonant peak will be measured than if purely collimated lightwere incident.

[0186] Because the lower sensitivity limit of a biosensor is related tothe ability to determine the peak maxima, it is important to measure anarrow resonant peak. Therefore, the use of a collimating illuminationsystem with the biosensor provides for the highest possible sensitivity.

[0187] One type of detection system for illuminating the biosensorsurface and for collecting the reflected light is a probe containing,for example, six illuminating optical fibers that are connected to alight source, and a single collecting optical fiber connected to aspectrometer. The number of fibers is not critical, any number ofilluminating or collecting fibers are possible. The fibers are arrangedin a bundle so that the collecting fiber is in the center of the bundle,and is surrounded by the six illuminating fibers. The tip of the fiberbundle is connected to a collimating lens that focuses the illuminationonto the surface of the biosensor.

[0188] In this probe arrangement, the illuminating and collecting fibersare side-by-side. Therefore, when the collimating lens is correctlyadjusted to focus light onto the biosensor surface, one observes sixclearly defined circular regions of illumination, and a central darkregion. Because the biosensor does not scatter light, but ratherreflects a collimated beam, no light is incident upon the collectingfiber, and no resonant signal is observed. Only by defocusing thecollimating lens until the six illumination regions overlap into thecentral region is any light reflected into the collecting fiber. Becauseonly defocused, slightly uncollimated light can produce a signal, thebiosensor is not illuminated with a single angle of incidence, but witha range of incident angles. The range of incident angles results in amixture of resonant wavelengths due to the dependence shown in FIG. 19.Thus, wider resonant peaks are measured than might otherwise bepossible.

[0189] Therefore, it is desirable for the illuminating and collectingfiber probes to spatially share the same optical path. Several methodscan be used to co-locate the illuminating and collecting optical paths.For example, a single illuminating fiber, which is connected at itsfirst end to a light source that directs light at the biosensor, and asingle collecting fiber, which is connected at its first end to adetector that detects light reflected from the biosensor, can each beconnected at their second ends to a third fiber probe that can act asboth an illuminator and a collector. The third fiber probe is orientedat a normal angle of incidence to the biosensor and supportscounter-propagating illuminating and reflecting optical signals. Anexample of such a detection system is shown in FIG. 18.

[0190] Another method of detection involves the use of a beam splitterthat enables a single illuminating fiber, which is connected to a lightsource, to be oriented at a 90 degree angle to a collecting fiber, whichis connected to a detector. Light is directed through the illuminatingfiber probe into the beam splitter, which directs light at thebiosensor. The reflected light is directed back into the beam splitter,which directs light into the collecting fiber probe. An example of sucha detection device is shown in FIG. 20. A beam splitter allows theilluminating light and the reflected light to share a common opticalpath between the beam splitter and the biosensor, so perfectlycollimated light can be used without defocusing.

[0191] Angular Scanning

[0192] Detection systems of the invention are based on collimated whitelight illumination of a biosensor surface and optical spectroscopymeasurement of the resonance peak of the reflected beam. Molecularbinding on the surface of a biosensor is indicated by a shift in thepeak wavelength value, while an increase in the wavelength correspondsto an increase in molecular absorption.

[0193] As shown in theoretical modeling and experimental data, theresonance peak wavelength is strongly dependent on the incident angle ofthe detection light beam. FIG. 19 depicts this dependence as modeled fora biosensor of the invention. Because of the angular dependence of theresonance peak wavelength, the incident white light needs to be wellcollimated. Angular dispersion of the light beam broadens the resonancepeak, and reduces biosensor detection sensitivity. In addition, thesignal quality from the spectroscopic measurement depends on the powerof the light source and the sensitivity of the detector. In order toobtain a high signal-to-noise ratio, an excessively long integrationtime for each detection location can be required, thus lengtheningoverall time to readout a biosensor plate. A tunable laser source can beused for detection of grating resonance, but is expensive.

[0194] In one embodiment of the invention, these disadvantages areaddressed by using a laser beam for illumination of a biosensor, and alight detector for measurement of reflected beam power. A scanningmirror device can be used for varying the incident angle of the laserbeam, and an optical system is used for maintaining collimation of theincident laser beam. See, e.g., “Optical Scanning” (Gerald F. Marchalled., Marcel Dekker (1991). Any type of laser scanning can be used. Forexample, a scanning device that can generate scan lines at a rate ofabout 2 lines to about 1,000 lines per second is useful in theinvention. In one embodiment of the invention, a scanning device scansfrom about 50 lines to about 300 lines per second.

[0195] In one embodiment, the reflected light beam passes through partof the laser scanning optical system, and is measured by a single lightdetector. The laser source can be a diode laser with a wavelength of,for example, 780 nm, 785 nm, 810 nm, or 830 nm. Laser diodes such asthese are readily available at power levels up to 150 mW, and theirwavelengths correspond to high sensitivity of Si photodiodes. Thedetector thus can be based on photodiode biosensors. An example of sucha detection system is shown in FIG. 52. A light source (100) provideslight to a scanner device (200), which directs the light into an opticalsystem (300) The optical system (300) directs light to a biosensor (400)Light is reflected from the biosensor (400) to the optical system (300),which then directs the light into a light signal detector (500). Oneembodiment of a detection system is shown in FIG. 21, which demonstratesthat while the scanning mirror changes its angular position, theincident angle of the laser beam on the surface changes by nominallytwice the mirror angular displacement. The scanning mirror device can bea linear galvanometer, operating at a frequency of about 2 Hz up toabout 120 Hz, and mechanical scan angle of about 10 degrees to about 20degrees. In this example, a single scan can be completed within about 10msec. A resonant galvanometer or a polygon scanner can also be used. Theexample shown in FIG. 21 includes a simple optical system for angularscanning. It consists of a pair of lenses with a common focal pointbetween them. The optical system can be designed to achieve optimizedperformance for laser collimation and collection of reflected lightbeam.

[0196] The angular resolution depends on the galvanometer specification,and reflected light sampling frequency. Assuming galvanometer resolutionof 30 arcsec mechanical, corresponding resolution for biosensor angularscan is 60 arcsec, i.e. 0.017 degree. In addition, assume a samplingrate of 100 ksamples/sec, and 20 degrees scan within 10 msec. As aresult, the quantization step is 20 degrees for 1000 samples, i.e. 0.02degree per sample. In this example, a resonance peak width of 0.2degree, as shown by Peng and Morris (Experimental demonstration ofresonant anomalies in diffraction from two-dimensional gratings, OpticsLett., 21:549 (1996)), will be covered by 10 data points, each of whichcorresponds to resolution of the detection system.

[0197] The advantages of such a detection system includes: excellentcollimation of incident light by a laser beam, high signal-to-noiseratio due to high beam power of a laser diode, low cost due to a singleelement light detector instead of a spectrometer, and high resolution ofresonance peak due to angular scanning.

[0198] Fiber Probe Biosensor

[0199] A biosensor of the invention can occur on the tip of a multi-modefiber optic probe. This fiber optic probe allows for in vivo detectionof biomarkers for diseases and conditions, such as, for example, cardiacartery disease, cancer, inflammation, and sepsis. A single biosensorelement (comprising, for example, several hundred grating periods) canbe fabricated into the tip of a fiber optic probe, or fabricated from aglass substrate and attached to the tip of a fiber optic probe. See FIG.17. A single fiber is used to provide illumination and measure resonantreflected signal.

[0200] For example, a fiber probe structure similar to that shown inFIG. 18 can be used to couple an illuminating fiber and detecting fiberinto a single counterpropagating fiber with a biosensor embedded orattached to its tip. The fiber optic probe is inserted into a mammalianbody, for example, a human body. Illumination and detection of areflected signal can occur while the probe is inserted in the body.

[0201] Mathematical Resonant Peak Determination

[0202] The sensitivity of a biosensor is determined by the shift in thelocation of the resonant peak when material is bound to the biosensorsurface. Because of noise inherent in the spectrum, it is preferable touse a procedure for determining an analytical curve—the turning point(i.e., peak) of which is well defined. Furthermore, the peakcorresponding to an analytic expression can be preferably determined togreater than sub-sampling-interval accuracy, providing even greatersensitivity.

[0203] One embodiment of the invention provides a method for determininga location of a resonant peak for a binding partner in a resonantreflectance spectrum with a colormetric resonant biosensor. The methodcomprises selecting a set of resonant reflectance data for a pluralityof colormetric resonant biosensors or a plurality of biosensor distinctlocations. The set of resonant reflectance data is collected byilluminating a colormetric resonant diffractive grating surface with alight source and measuring reflected light at a pre-determinedincidence. The colormetric resonant diffractive grating surface is usedas a surface binding platform for one or more specific bindingsubstances such that binding partners can be detected without use of amolecular label.

[0204] The step of selecting a set of resonant reflectance data caninclude selecting a set of resonant reflectance data:

x _(i)andy _(i)fori=1,2,3, . . . n,

[0205] wherein x_(i) is where a first measurement includes a firstreflectance spectra of one or more specific binding substances attachedto the colormetric resonant diffractive grating surface, y_(i) and asecond measurement and includes a second reflectance spectra of the oneor more specific binding substances after a plurality of bindingpartners are applied to colormetric resonant diffractive grating surfaceincluding the one or more specific binding substances, and n is a totalnumber of measurements collected.

[0206] The set of resonant reflectance data includes a plurality of setsof two measurements, where a first measurement includes a firstreflectance spectra of one or more specific binding substances that areattached to the colormetric resonant diffractive grating surface and asecond measurement includes a second reflectance spectra of the one ormore specific binding substances after one or more binding partners areapplied to the colormetric resonant diffractive grating surfaceincluding the one or more specific binding substances. A difference in apeak wavelength between the first and second measurement is ameasurement of an amount of binding partners that bound to the one ormore specific binding substances. A sensitivity of a colormetricresonant biosensor can be determined by a shift in a location of aresonant peak in the plurality of sets of two measurements in the set ofresonant reflectance data.

[0207] A maximum value for a second measurement from the plurality ofsets of two measurements is determined from the set of resonantreflectance data for the plurality of binding partners, wherein themaximum value includes inherent noise included in the resonantreflectance data. A maximum value for a second measurement can includedetermining a maximum value y_(k) such that:

(y _(k) >=y _(i)) for alli≠k.

[0208] It is determined whether the maximum value is greater than apre-determined threshold. This can be calculated by, for example,computing a mean of the set of resonant reflectance data; computing astandard deviation of the set of resonant reflectance data; anddetermining whether ((y_(k)−mean)/standard deviation) is greater than apre-determined threshold. The pre-determined threshold is determined bythe user. The user will determine what amount of sensitivity is desiredand will set the pre-determined threshold accordingly.

[0209] If the maximum value is greater than a pre-determined threshold acurve-fit region around the determined maximum value is defined. Thestep of defining a curve-fit region around the determined maximum valuecan include, for example:

[0210] defining a curve-fit region of (2w+1) bins, wherein w is apre-determined accuracy value;

[0211] extracting (x_(i), k−w<=i<=k+w); and

[0212] extracting (y₁, k−w<=i<=k+w). A curve-fitting procedure isperformed to fit a curve around the curve-fit region, wherein thecurve-fitting procedure removes a pre-determined amount of inherentnoise included in the resonant reflectance data. A curve-fittingprocedure can include, for example:

[0213] computing g_(i)=1n y_(i);

[0214] performing a 2^(nd) order polynomial fit on g_(i) to obtaing′_(i) defined on (x₁, k−w<=i<=k+w);

[0215] determining from the 2^(nd) order polynomial fit coefficients a,b and c of for (ax² +bx+c)−; and

[0216] computing y′_(i)=e^(g′i).

[0217] The location of a maximum resonant peak is determined on thefitted curve, which can include, for example, determining a location ofmaximum reasonant peak (x_(p)=(−b)/2a). A value of the maximum resonantpeak is determined, wherein the value of the maximum resonant peak isused to identify an amount of biomolecular binding of the one or morespecific binding substances to the one or more binding partners. A valueof the maximum resonant peak can include, for example, determining thevalue with of x_(p) at y′_(p).

[0218] One embodiment of the invention comprises a computer readablemedium having stored therein instructions for causing a processor toexecute a method for determining a location of a resonant peak for abinding partner in a resonant reflectance spectrum with a colormetricresonant biosensor. A computer readable medium can include, for example,magnetic disks, optical disks, organic memory, and any other volatile(e.g., Random Access Memory (“RAM”)) or non-volatile (e.g., Read-OnlyMemory (“ROM”)) mass storage system readable by the processor. Thecomputer readable medium includes cooperating or interconnected computerreadable medium, which exist exclusively on a processing system or to bedistributed among multiple interconnected processing systems that can belocal or remote to the processing system.

[0219] The following are provided for exemplification purpose only andare not intended to limit the scope of the invention described in broadterms above. All references cited in this disclosure are incorporatedherein by reference.

EXAMPLE 1

[0220] Fabrication of a SWS Biosensor

[0221] An example of biosensor fabrication begins with a flat glasssubstrate that is coated with a thin layer (180 nm) of silicon nitrideby plasma-enhanced chemical vapor deposition (PECVD).

[0222] The desired structure is first produced in photoresist bycoherently exposing a thin photoresist film to three laser beams, asdescribed in previously (Cowen, “The recording and large scalereplication of crossed holographic grating arrays using multiple beaminterferometry,” in International Conference on the Application, Theory,and Fabrication of Periodic Structures, Diffraction Gratings, and MoirePhenomena II, J. M. Lemer, ed., Proc. Soc. Photo-Opt. Instrum. Eng.,503, 120-129, 1984; Cowen, “Holographic honeycomb microlens,” Opt. Eng.24, 796-802 (1985); Cowen & Slafer, “The recording and replication ofholographic micropatterns for the ordering of photographic emulsiongrains in film systems,” J. Imaging Sci. 31, 100-107, 1987. Thenonlinear etching characteristics of photoresist are used to develop theexposed film to create a pattern of holes within a hexagonal grid, asshown in FIG. 22. The photoresist pattern is transferred into thesilicon nitride layer using reactive ion etching (RIE). The photoresistis removed, and a cover layer of spin-on-glass (SOG) is applied(Honeywell Electronic Materials, Sunnyvale, Calif.) to fill in the openregions of the silicon nitride grating. The structure of the top surfaceof the finished biosensor is shown in FIG. 23. A photograph of finishedparts are shown in FIG. 24.

EXAMPLE 2

[0223] A SRVD biosensor was prepared by making five circular diffusegrating holograms by stamping a metal master plate into vinyl. Thecircular holograms were cut out and glued to glass slides. The slideswere coated with 1000 angstroms of aluminum. In air, the resonantwavelength of the grating is ˜380 nm, and therefore, no reflected coloris visible. When the grating is covered with water, a light bluereflection is observed. Reflected wavelength shifts are observable andmeasurable while the grating is covered with a liquid, or if a specificbinding substances and/or binding partners cover the structure.

[0224] Both proteins and bacteria were immobilized onto the surface of aSRVD biosensor at high concentration and the wavelength shift wasmeasured. For each material, a 20 droplet is placed onto a biosensordistinct location and allowed to dry in air. At 1 g/ml proteinconcentration, a 20 droplet spreads out to cover a 1 cm diameter circleand deposits about 2×10⁻⁸ grams of material. The surface density is 25.6ng/mm².

[0225] For high concentration protein immobilization (biosensor 4) a 10droplet of 0.8 g bovine serum albumin (BSA) in 40 ml DI H₂O is spreadout to cover a 1 cm diameter circle on the surface of a biosensor. Thedroplet deposits 0.0002 g of BSA, for a density of 2.5e-6 g/mm². Afterprotein deposition, biosensor 4 has a green resonance in air.

[0226] For bacteria immobilization (biosensor 2) a 20 droplet of NECKborrelia Lyme Disease bacteria (1.8e8 cfu/ml) was deposited on thesurface of a biosensor. After bacteria deposition, the biosensor looksgrey in air.

[0227] For low concentration protein immobilization (biosensor 6) a 10droplet of 0.02% of BSA in DI H₂O (0.8 g BSA in 40 ml DI H₂O) is spreadout to cover a 1 cm diameter circle. The droplet deposits 0.000002 g ofBSA for a density of 2.5e-8 g/mm². After protein deposition, biosensor 6looks grey in air.

[0228] In order to obtain quantitative data on the extent of surfacemodification resulting from the above treatments, the biosensors weremeasured using a spectrometer.

[0229] Because a green resonance signal was immediately visuallyobserved on the biosensor upon which high concentration BSA wasdeposited (biosensor 4), it was measured in air. FIG. 25 shows two peaksat 540 nm and 550 nm in green wavelengths where none were present beforeprotein deposition, indicating that the presence of a protein thin filmis sufficient to result in a strong shift in resonant wavelength of asurface relief structure.

[0230] Because no visible resonant wavelength was observed in air forthe slide upon which a low concentration of protein was applied(biosensor 6), it was measured with distilled water on surface andcompared against a biosensor which had no protein treatment. FIG. 26shows that the resonant wavelength for the slide with protein appliedshifted to green compared to a water-coated slide that had not beentreated.

[0231] Finally, a water droplet containing Lyme Disease bacteriaBorrelia burgdorferi was applied to a grating structure and allowed todry in air (biosensor 2). Because no visually observed resonanceoccurred in air after bacteria deposition, the biosensor was measuredwith distilled water on the surface and compared to a water-coatedbiosensor that had undergone no other treatment. As shown in FIG. 27,the application of bacteria results in a resonant frequency shift tolonger wavelengths.

EXAMPLE 3

[0232] Computer Model of Biosensor

[0233] To demonstrate the concept that a resonant grating structure canbe used as a biosensor by measuring the reflected wavelength shift thatis induced when biological material is adsorbed onto its surface, thestructure shown in FIG. 1 was modeled by computer. For purposes ofdemonstration, the substrate chosen was glass (n_(substrate=)1.50). Thegrating is a two-dimensional pattern of silicon nitride squares (t₂=180nm, n₂=2.01, k₂=0.001) with a period of 510 nm, and a filling factor of56.2% (i.e. 56.2% of the surface is covered with silicon nitride squareswhile the rest is the area between the squares). The areas betweensilicon nitride squares are filled with a lower refractive indexmaterial. The same material also covers the squares and provides auniformly flat upper surface. For this simulation, a glass layer wasselected (n₁=1.40) that covers the silicon nitride squares by t₂=100 nm.To observe the effect on the reflected wavelength of this structure withthe deposition of biological material, variable thicknesses of protein(n_(bio)=1.5) were added above the glass coating layer.

[0234] The reflected intensity as a function of wavelength was modeledusing GSOLVER software, which utilizes full 3-dimensional vector codeusing hybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination may be from any incidence and any polarization.

[0235] The results of the computer simulation are shown in FIG. 28 andFIG. 29. As shown in FIG. 28, the resonant structure allows only asingle wavelength, near 780 nm, to be reflected from the surface when noprotein is present on the surface. Because the peak width athalf-maximum is ˜1.5 nm, resonant wavelength shifts of ˜0.2 nm will beeasily resolved. FIG. 28 also shows that the resonant wavelength shiftsto longer wavelengths as more protein is deposited on the surface of thestructure. Protein thickness changes of 2 nm are easily observed. FIG.29 plots the dependence of resonant wavelength on the protein coatingthickness. A near linear relationship between protein thickness andresonant wavelength is observed, indicating that this method ofmeasuring protein adsorption can provide quantitative data. For thesimulated structure, FIG. 29 shows that the wavelength shift responsebecomes saturated when the total deposited protein layer exceeds ˜250nm. This upper limit for detection of deposited material providesadequate dynamic range for any type of biomolecular assay.

EXAMPLE 4

[0236] Computer Model of Biosensor

[0237] In another embodiment of the invention a biosensor structureshown in FIG. 30 was modeled by computer. For purposes of demonstration,the substrate chosen was glass n_(substrate)=1.454 coated with a layerof high refractive index material such as silicon nitride, zinc sulfide,tantalum oxide, or titanium dioxide. In this case, silicon nitride(t₃=90 nm, n₃=2.02) was used. The grating is two-dimensional pattern ofphotoresist squares (t₂=90 nm, n₂=1.625 ) with a period of 510 nm, and afilling factor of 56.2% (i.e. 56.2% of the surface is covered withphotoresist squares while the rest is the area between the squares). Theareas between photoresist squares are filled with a lower refractiveindex material such as glass, plastic, or epoxy. The same material alsocovers the squares and provides a uniformly flat upper surface. For thissimulation, a glass layer was selected (n₁=1.45 ) that covers thephotoresist squares by t₂=100 nm. To observe the effect on the reflectedwavelength of this structure with the deposition of a specific bindingsubstance, variable thicknesses of protein (n_(bio)=1.5 ) were addedabove the glass coating layer.

[0238] The reflected intensity as a function of wavelength was modeledusing GSOLVER software, which utilizes full 3-dimensional vector codeusing hybrid Rigorous Coupled Wave Analysis and Modal analysis. GSOLVERcalculates diffracted fields and diffraction efficiencies from planewave illumination of arbitrarily complex grating structures. Theillumination may be from any incidence and any polarization.

[0239] The results of the computer simulation are shown in FIG. 31 andFIG. 32. The resonant structure allows only a single wavelength, near805 nm, to be reflected from the surface when no protein is present onthe surface. Because the peak width at half-maximum is <0.25 nm,resonant wavelength shifts of 1.0 nm will be easily resolved. FIG. 31also shows that the resonant wavelength shifts to longer wavelengths asmore protein is deposited on the surface of the structure. Proteinthickness changes of 1 nm are easily observed. FIG. 32 plots thedependence of resonant wavelength on the protein coating thickness. Anear linear relationship between protein thickness and resonantwavelength is observed, indicating that this method of measuring proteinadsorption can provide quantitative data.

EXAMPLE 5

[0240] Sensor Readout Instrumentation

[0241] In order to detect reflected resonance, a white light source canilluminate a ˜1 mm diameter region of a biosensor surface through a 400micrometer diameter fiber optic and a collimating lens, as shown in FIG.33. Smaller or larger areas may be sampled through the use ofillumination apertures and different lenses. A group of six detectionfibers are bundled around the illumination fiber for gathering reflectedlight for analysis with a spectrometer (Ocean Optics, Dunedin, Fla.).For example, a spectrometer can be centered at a wavelength of 800 nm,with a resolution of ˜0.14 nm between sampling bins. The spectrometerintegrates reflected signal for 25-75 msec for each measurement. Thebiosensor sits upon an x-y motion stage so that different regions of thebiosensor surface can be addressed in sequence.

[0242] Equivalent measurements can be made by either illuminating thetop surface of device, or by illuminating through the bottom surface ofthe transparent substrate. Illumination through the back is preferredwhen the biosensor surface is immersed in liquid, and is most compatiblewith measurement of the biosensor when it is incorporated into thebottom surface of, for example, a microwell plate.

EXAMPLE 6

[0243] Demonstration of Resonant Reflection

[0244]FIG. 34 shows the resonant reflectance spectra taken from abiosensor as shown in FIG. 1 using the instrumentation described inExample 5. The wavelength of the resonance (λ_(peak)=772.5 nm) compareswith the resonant wavelength predicted by the computer model(λ_(peak)=781 nm), and the measured reflectance efficiency (51%) iscomparable to the predicted efficiency (70%). The greatest discrepancybetween the measured and predicted characteristics is the linewidth ofthe resonant peak. The measured full-width at half maximum (FWHM) of theresonance is 6 nm, while the predicted FWHM is 1.5 nm. As will be shown,the dominant source of the larger measured FWHM is collimation of theillumination optics, which can easily be corrected.

[0245] As a basic demonstration of the resonant structure's ability todetect differences in the refractive index of materials in contact withits surface, a biosensor was exposed to a series of liquids withwell-characterized optical properties. The liquids used were water,methanol, isopropyl alcohol, acetone, and DMF. A biosensor was placedface-down in a small droplet of each liquid, and the resonant wavelengthwas measured with a fiber illumination/detection probe facing thebiosensor's back side. Table 1 shows the calculated and measured peakresonant wavelength as a biosensor surface is exposed to liquids withvariable refractive index demonstrating the correlation between measuredand theoretical detection sensitivity. As shown in Table 1, the measuredresonant peak positions and measured resonant wavelength shifts arenearly identical to the predicted values. This example demonstrates theunderlying sensitivity of the biosensor, and validates the computermodel that predicts the wavelength shift due to changes in the materialin contact with the surface. TABLE 1 Calculated Measured Peak PeakWavelength Wavelength Solution n (nm) Shift (nm) (nm) Shift (nm) Water1.333  791.6 0 786.08 0 Isopropyl 1.3776 795.9 4.3 789.35 3.27 Acetone1.3588 794 2.4 788.22 2.14 Methanol 1.3288 791.2 −0.4 785.23 −0.85 DMF1.4305 802 10.4 796.41 10.33

[0246] Similarly, a biosensor is able to measure the refractive indexdifference between various buffer solutions. As an example, FIG. 35shows the variation in peak wavelength with the concentration of bovineserum albumin (BSA) in water. Resonance was measured with the biosensorplaced face-down in a droplet of buffer, and rinsed with water betweeneach measurement.

EXAMPLE 7

[0247] Immobilized Protein Detection

[0248] While the detection experiments shown in Example 6 demonstrate abiosensor's ability to measure small differences in refractive index ofliquid solutions, the biosensor is intended to measure specific bindingsubstances and binding partners that are chemically bound to thebiosensor surface. In order to demonstrate a biosensor's ability toquantify biomolecules on its surface, droplets of BSA dissolved in PBSat various concentrations were applied to a biosensor as shown inFIG. 1. The 3 μl droplets were allowed to dry in air, leaving a smallquantity of BSA distributed over a ˜2 mm diameter area. The peakresonant wavelength of each biosensor location was measured before andafter droplet deposition, and the peak wavelength shift was recorded.See FIG. 37.

EXAMPLE 8

[0249] Immobilization of One or More Specific Binding Substances

[0250] The following protocol was used on a colorimetric resonantreflective biosensor to activate the surface with amine functionalgroups. Amine groups can be used as a general-purpose surface forsubsequent covalent binding of several types of linker molecules.

[0251] A biosensor of the invention is cleaned by immersing it intopiranha etch (70/30% (v/v) concentrated sulfuric acid /30% hydrogenperoxide) for 12 hours. The biosensor was washed thoroughly with water.The biosensor was dipped in 3% 3-aminopropyltriethoxysilane solution indry acetone for 1 minute and then rinsed with dry acetone and air-dried.The biosensor was then washed with water.

[0252] A semi-quantitative method is used to verify the presence ofamino groups on the biosensor surface. One biosensor from each batch ofamino-functionalized biosensors is washed briefly with 5 mL of 50 mMsodium bicarbonate, pH 8.5. The biosensor is then dipped in 5 mL of 50mM sodium bicarbonate, pH 8.5 containing 0.1 mMsulfo-succinimidyl-4-O-(4,4′-dimethoxytrityl)-butyrate (s-SDTB, Pierce,Rockford, Ill.) and shaken vigorously for 30 minutes. The s-SDTBsolution is prepared by dissolving 3.0 mg of s-SDTB in 1 mL of DMF anddiluting to 50 mL with 50 mM sodium bicarbonate, pH 8.5. After a 30minute incubation, the biosensor is washed three times with 20 mL ofddH2O and subsequently treated with 5 mL 30% perchloric acid. Thedevelopment of an orange-colored solution indicates that the biosensorhas been successfully derivatized with amines; no color change isobserved for untreated glass biosensors.

[0253] The absorbance at 495nm of the solution after perchloric acidtreatment following the above procedure can be used as an indicator ofthe quantity of amine groups on the surface. In one set of experiment,the absorbance was 0.627, 0.647, and 0.728 for Sigma slides,Cel-Associate slides, and in-house biosensor slides, respectively. Thisindicates that the level of NH₂ O activation of the biosensor surface iscomparable in the activation commercially available microarray glassslides.

[0254] After following the above protocol for activating the biosensorwith amine, a linker molecule can be attached to the biosensor. Whenselecting a cross-linking reagent, issues such as selectivity of thereactive groups, spacer arm length, solubility, and cleavability shouldbe considered. The linker molecule, in turn, binds the specific bindingsubstance that is used for specific recognition of a binding partner. Asan example, the protocol below has been used to bind a biotin linkermolecule to the amine-activated biosensor.

[0255] Protocol for Activating Amine-Coated Biosensor with Biotin

[0256] Wash an amine-coated biosensor with PBS (pH 8.0) three times.Prepare sulfo-succinimidyl-6-(biotinamido)hexanoate(sulfo-NHS-LC-biotin, Pierce, Rockford, Ill.) solution in PBS buffer (pH8) at 0.5 mg/ml concentration. Add 2 ml of the sulfo-NHS-LC-biotinsolution to each amine-coated biosensor and incubate at room temperaturefor 30 min. Wash the biosensor three times with PBS (pH 8.0). Thesulfo-NHS-LC-biotin linker has a molecular weight of 556.58 and a lengthof 22.4 Å. The resulting biosensors can be used for capturing avidin orstrepavidin molecules.

[0257] Protocol for Activatinz Amine-Coated Biosensor with Aldehyde

[0258] Prepare 2.5% glutaraldehyde solution in 0.1 M sodium phosphate,0.05% sodium azide, 0.1% sodium cyanoborohydride, pH 7.0. Add 2 ml ofthe sulfo-NHS-LC-biotin solution to each amine-coated biosensor andincubate at room temperature for 30 min. Wash the biosensor three timeswith PBS (pH 7.0). The glutaraldehyde linker has a molecular weight of100.11. The resulting biosensors can be used for binding proteins andother amine-containing molecules. The reaction proceeds through theformation of Schiff bases, and subsequent reductive animation yieldsstable secondary amine linkages. In one experiment, where a coatedaldehyde slide made by the inventors was compared to a commerciallyavailable aldehyde slide (Cel-Associate), ten times higher binding ofstreptavidin and anti-rabbit IgG on the slide made by the inventors wasobserved.

[0259] Protocol for Activating Amine-coated Biosensor with NHS

[0260] 25 mM N,N′-disuccinimidyl carbonate (DSC, Sigma Chemical Company,St. Louis, Miss.) in sodium carbonate buffer (pH 8.5) was prepared. 2 mlof the DSC solution was added to each amine-coated biosensor andincubated at room temperature for 2 hours. The biosensors were washedthree times with PBS (pH 8.5). A DSC linker has a molecular weight of256.17. Resulting biosensors are used for binding to hydroxyl- oramine-containing molecules. This linker is one of the smallesthomobifunctional NHS ester cross-linking reagents available.

[0261] In addition to the protocols defined above, many additionalsurface activation and molecular linker techniques have been reportedthat optimize assay performance for different types of biomolecules.Most common of these are amine surfaces, aldehyde surfaces, and nickelsurfaces. The activated surfaces, in turn, can be used to attach severaldifferent types of chemical linkers to the biosensor surface, as shownin Table 2. While the amine surface is used to attach several types oflinker molecules, the aldehyde surface is used to bind proteinsdirectly, without an additional linker. A nickel surface is usedexclusively to bind molecules that have an incorporated histidine(“his”) tag. Detection of “his-tagged” molecules with a Nickel activatedsurface is well known (Sigal et al., Anal. Chem. 68, 490 (1996)).

[0262] Table 2 demonstrates an example of the sequence of steps that areused to prepare and use a biosensor, and various options that areavailable for surface activation chemistry, chemical linker molecules,specific binding substances and binding partners molecules.Opportunities also exist for enhancing detected signal throughamplification with larger molecules such as HRP or streptavidin and theuse of polymer materials such as dextran or TSPS to increase surfacearea available for molecular binding. TABLE 2 Label Bare Surface LinkerReceptor Detected Molecule Sensor Activation Molecule Molecule Material(Optional) Glass Amino SMPT Sm m'cules Peptide Enhance sensitivityPolymers NHS-Biotin Peptide Med Protein 1000x optional to Aldehyde DMPMed Protein Lrg Protein HRP enhance .IgG sensitivity NNDC Lrg ProteinStreptavidin 2-5x Ni .IgG Phage Dextran His-tag Cell TSPS Others . . .cDNA cDNA

EXAMPLE 9

[0263] IgG Assay

[0264] As an initial demonstration for detection of biochemical binding,an assay was performed in which a biosensor was prepared by activationwith the amino surface chemistry described in Example 8 followed byattachment of a biotin linker molecule. The biotin linker is used tocovalently bond a streptavidin receptor molecule to the surface byexposure to a 50 μg/ml concentration solution of streptavidin in PBS atroom temperature for 2-4 hours. The streptavidin receptor is capable ofbinding any biotinylated protein to the biosensor surface. For thisexample, 3 μl droplets of biotinylated anti-human IgG in phosphatebuffer solution (PBS) were deposited onto 4 separate locations on thebiosensor surface at a concentration of 200 μg/ml. The solution wasallowed to incubate on the biosensor for 60 min before rinsingthoroughly with PBS. The peak resonant wavelength of the 4 locationswere measured after biotin activation, after streptavidin receptorapplication, and after ah-IgG binding. FIG. 37 shows that the additionof streptavidin and ah-IgG both yield a clearly measurable increase inthe resonant wavelength.

EXAMPLE 10

[0265] Biotin/Streptavidin Assay

[0266] A series of assays were performed to detect streptavidin bindingby a biotin receptor layer. A biosensor was first activated with aminochemistry, followed by attachment of a NHS-Biotin linker layer, aspreviously described. Next, 3 μdroplets of streptavidin in PBS wereapplied to the biosensor at various concentrations. The droplets wereallowed to incubate on the biosensor surface for 30 min beforethoroughly washing with PBS rinsing with DI water. The peak resonantwavelength was measured before and after streptavidin binding, and theresonant wavelength shifts are shown in FIG. 38. A linear relationshipbetween peak wavelength and streptavidin concentration was observed, andin this case the lowest streptavidin concentration measured was 0.2μg/ml. This concentration corresponds to a molarity of 3.3 nM.

EXAMPLE 11

[0267] Protein-Protein Binding Assay

[0268] An assay was performed to demonstrate detection ofprotein-protein interactions. As described previously, a biosensor wasactivated with amino chemistry and an NHS-biotin linker layer. A goatanti-biotin antibody receptor layer was attached to the biotin linker byexposing the biosensor to a 50 μg/ml concentration solution in PBS for60 min at room temperature followed by washing in PBS and rinsing withDI water. In order to prevent interaction of nonspecific proteins withunbound biotin on the biosensor surface, the biosensor surface wasexposed to a 1% solution of bovine serum albumin (BSA) in PBS for 30min. The intent of this step is to “block” unwanted proteins frominteracting with the biosensor. As shown in FIG. 39 a significant amountof BSA is incorporated into the receptor layer, as shown by the increasein peak wavelength that is induced. Following blocking, 3 μl droplets ofvarious concentrations of anti-goat IgG were applied to separatelocations on the biosensor surface. The droplets were allowed toincubate for 30 min before thorough rinsing with DI water. The biosensorpeak resonant wavelength was measured before blocking, after blocking,after receptor layer binding, and after anti-goat IgG detection for eachspot. FIG. 39 shows that an anti-goat IgG concentration of 10 μg/mlyields an easily measurable wavelength shift.

EXAMPLE 12

[0269] Unlabeled ELISA Assay

[0270] Another application of a biosensor array platform is its abilityto perform Enzyme-Linked Immunosorbent Assays (ELISA) without the needfor an enzyme label, and subsequent interaction an enzyme-specificsubstrate to generate a colored dye. FIG. 40 shows the results of anexperiment where a biosensor was prepared to detect interferon-γ (IFN-γ)with an IFN-γ antibody receptor molecule. The receptor molecule wascovalently attached to an NH₂-activated biosensor surface with an SMPTlinker molecule (Pierce Chemical Company, Rockford, Ill.). The peakresonant wavelength shift for application of the NH₂, SMPT, andanti-human IFN-α receptor molecules were measured for two adjacentlocations on the biosensor surface, as shown in FIG. 40. The twolocations were exposed to two different protein solutions in PBS at aconcentration of 100 μg/ml. The first location was exposed to IFN-γ,which is expected to bind with the receptor molecule, while the secondwas exposed to neural growth factor (NGF), which is not expected to bindwith the receptor. Following a 30 minute incubation the biosensor wasmeasured by illuminating from the bottom, while the top surface remainedimmersed in liquid. The location exposed to IFN-γ registered awavelength shift of 0.29 nm, while the location exposed to NGFregistered a wavelength shift of only 0.14 mn. Therefore, without theuse of any type of enzyme label or color-generating enzyme reaction, thebiosensor was able to discriminate between solutions containingdifferent types of protein.

EXAMPLE 13

[0271] Protease Inhibitor Assay (Caspase-3)

[0272] A Caspase-3 protease inhibitor assay was performed to demonstratethe biosensor's ability to measure the presence and cleavage of smallmolecules in an experimental context that is relevant to pharmaceuticalcompound screening.

[0273] Caspases (Cysteine-requiring Aspartate protease) are a family ofproteases that mediate cell death and are important in the process ofapoptosis. Caspase 3, an effector caspase, is the most studied ofmammalian caspases because it can specifically cleave most knowncaspase-related substrates. The caspase 3 assay is based on thehydrolysis of the 4-amino acid peptide substrate NHS-Gly-Asp-Glu-Val-Aspp-nitroanilide (NHS-GDEVD-pNA) by caspase 3, resulting in the release ofthe pNA moiety.${\left( {{NHS} - {GDEVD} - {pNA}} \right)\overset{{Caspase}\quad 3}{}\left( {{NHS} - {GDEVD}} \right)} + {pNA}$

[0274] The NHS molecule attached to the N-terminal of the GDEVD providesa reactive end group to enable the NHS-GDEVD-pNA complex to becovalently bound to the biosensor with the pNA portion of the complexoriented away from the surface. Attached in this way, the caspase-3 willhave the best access to its substrate cleavage site.

[0275] A biosensor was prepared by cleaning in 3:1 H₂SO₄:H₂O₂ solution(room temperature, 1 hour), followed by silanation (2% silane in dryacetone, 30 sec) and attachment of a poly-phe-lysine (PPL) layer (100μg/ml PPL in PBS pH 6.0 with 0.5 M NaCl, 10 hours). The NHS-GDEVD-pNAcomplex was attached by exposing the biosensor to a 10 mM solution inPBS (pH 8.0, room temperature, 1 hour). A microwell chamber was sealedover the biosensor surface, and cleavage of pNA was performed byaddition of 100 μl of caspase-3 in 1×enzyme buffer (100 ng/ml, roomtemperature, 90 minutes). Following exposure to the caspase 3 solution,the biosensor was washed in PBS. A separate set of experiments using aspectrophotometer were used to confirm the attachment of the complex tothe surface of the biosensor, and functional activity of the caspase-3for removal of the pNA molecule from the surface-bound complex.

[0276] The peak resonant frequency of the biosensor was measured beforeattachment of the NHS-GDEVD-pNA complex, after attachment of the complex(MW=860 Da), and after cleavage of the pNA (MW=136) with caspase 3. Asshown in FIG. 41, the attachment of the peptide molecule is clearlymeasurable, as is the subsequent removal of the pNA. The pNA removalsignal of Δλ=0.016 nm is 5.3×higher than the minimum detectable peakwavelength shift of 0.003 nm. The proportion of the added molecularweight and subtracted molecular weight (860 Da/136 Da=6.32) are in closeagreement with the proportion of peak wavelength shift observed for theadded and subtracted material (0.082 nm /0.016 nm=5.14).

[0277] The results of this experiment confirm that a biosensor iscapable of measuring small peptides (in this case, a 5-mer peptide)without labels, and even detecting the removal of 130 Da portions of amolecule through the activity of an enzyme.

EXAMPLE 14

[0278] Reaction Kinetics for Protein-Protein Binding Assays

[0279] Because a biosensor of the invention can be queried continuouslyas a function of time while it is immersed in liquid, a biosensor can beutilized to perform both endpoint-detection experiments and to obtainkinetic information about biochemical reactions. As an example, FIG. 42shows the results of an experiment in which a single biosensor locationis measured continuously through the course of consecutively addingvarious binding partners to the surface. Throughout the experiment, adetection probe illuminated the biosensor through the back of thebiosensor substrate, while biochemistry is performed on the top surfaceof the device. A rubber gasket was sealed around the measured biosensorlocation so that added reagents would be confined, and all measurementswere performed while the top surface of the biosensor was immersed inbuffer solution. After initial cleaning, the biosensor was activatedwith NH₂, and an NHS-Biotin linker molecule. As shown in FIG. 42, goatα-biotin antibodies of several different concentrations (1, 10, 100,1000 μg/ml) were consecutively added to the biosensor and allowed toincubate for 30 minutes while the peak resonant wavelength wasmonitored. Following application of the highest concentration α-BiotinIgG, a second layer of protein was bound to the biosensor surfacethrough the addition of α-goat IgG at several concentrations (0.1, 1,10, and 100 μg/ml). Again, the resonant peak was continuously monitoredas each solution was allowed to incubate on the biosensor for 30minutes. FIG. 42 shows how the resonant peak shifted to greaterwavelength at the end of each incubation period.

[0280]FIG. 43 shows the kinetic binding curve for the final resonantpeak transitions from FIG. 42, in which 100 μg/ml of cc-goat IgG isadded to the biosensor. The curve displays the type of profile that istypically observed for kinetic binding experiments, in which a rapidincrease from the base frequency is initially observed, followed by agradual saturation of the response. This type of reaction profile wasobserved for all the transitions measured in the experiment. FIG. 44shows the kinetic binding measurement of IgG binding.

[0281] The removal of material from the biosensor surface through theactivity of an enzyme is also easily observed. When the biosensor fromthe above experiment (with two protein coatings of goat anti-biotin IgGand anti-goat IgG) is exposed to the protease pepsin at a concentrationof 1 mg/ml, the enzyme dissociates both IgG molecules, and removes themfrom the biosensor surface. As shown in FIG. 45, the removal of boundmolecules from the surface can be observed as a function of time.

EXAMPLE 15

[0282] Proteomics Applications

[0283] Biosensors of the invention can be used for proteomicsapplications. A biosensor array can be exposed to a test sample thatcontains a mixture of binding partners comprising, for example, proteinsor a phage display library, and then the biosensor surface is rinsed toremove all unbound material. The biosensor is optically probed todetermine which distinct locations on the biosensor surface haveexperienced the greatest degree of binding, and to provide aquantitative measure of bound material. Next, the biosensor is placed ina “flow cell” that allows a small (e.g., <50 microliters) fixed volumeof fluid to make contact to the biosensor surface. One electrode isactivated so as to elute bound material from only a selected biosensorarray distinct location. The bound material becomes diluted within theflow cell liquid. The flow cell liquid is pumped away from the biosensorsurface and is stored within a microtiter plate or some other container.The flow cell liquid is replaced with fresh solution, and a newbiosensor electrode is activated to elute its bound binding partners.The process is repeated until all biosensor distinct locations ofinterest have been eluted and gathered into separate containers. If thetest sample liquid contained a mixture of proteins, protein contentswithin the separate containers can be analyzed using a technique such aselectrospray tandem mass spectrometry. If the sample liquid contained aphage display library, the phage clones within the separate containerscan be identified through incubation with a host strain bacteria,concentration amplification, and analysis of the relevant library DNAsequence.

EXAMPLE 16

[0284] Mathematical Resonant Peak Determination

[0285] This example discusses some of the findings that have beenobtained from looking at fitting different types of curves to theobserved data.

[0286] The first analytic curve examined is a second-order polynomial,given by

y=ax ² +bx+c

[0287] The least-squares solution to this equation is given by the costfunction${\varphi = {\sum\limits_{i = 1}^{n}\quad \left( {{ax}_{i}^{2} + {bx}_{i} + c - y_{i}} \right)^{2}}},$

[0288] the minimization of which is imposed by the constraints$\frac{\partial\varphi}{\partial a} = {\frac{\partial\varphi}{\partial b} = {\frac{\partial\varphi}{\partial c} = 0.}}$

[0289] Solving these constraints for a, b, and c yields $\begin{pmatrix}a \\b \\c\end{pmatrix} = {\begin{pmatrix}{\sum x_{i}^{4}} & {\sum x_{i}^{3}} & {\sum x_{i}^{2}} \\{\sum x_{i}^{3}} & {\sum x_{i}^{2}} & {\sum x_{i}} \\{\sum{x\quad 2}} & {\sum x_{i}} & n\end{pmatrix}^{- 1} \cdot {\begin{pmatrix}{\sum{x_{i}^{2}y_{i}}} \\{\sum{x_{i}y_{i}}} \\{\sum y_{i}}\end{pmatrix}.}}$

[0290] The result of one such fit is shown in FIG. 46; the acquired dataare shown as dots and the 2^(nd)-order polynomial curve fit is shown asthe solid line.

[0291] Empirically, the fitted curve does not appear to have sufficientrise and fall near the peak. An analytic curve that provides bettercharacteristics in this regard is the exponential, such as a Gaussian. Asimple method for performing a Gaussian-like fit is to assume that theform of the curve is given by

y=e ^(ax) ² _(+bx+c),

[0292] in which case the quadratic equations above can be utilized byforming y′, where y′=1ny. FIG. 46 shows the result of such a fit. Thevisual appearance of FIG. 46 indicates that the exponential is a betterfit, providing a 20% improvement over that of the quadratic fit.

[0293] Assuming that the exponential curve is the preferred data fittingmethod, the robustness of the curve fit is examined in two ways: withrespect to shifts in the wavelength and with respect to errors in thesignal amplitude.

[0294] To examine the sensitivity of the analytical peak location whenthe window from which the curve fitting is performed is altered to fall10 sampling intervals to the left or to the right of the true maxima.The resulting shift in mathematically-determined peak location is shownin Table 3. The conclusion to be derived is that the peak location isreasonably robust with respect to the particular window chosen: for ashift of ˜1.5 nm, the corresponding peak location changed by only <0.06nm, or 4 parts in one hundred sensitivity.

[0295] To examine the sensitivity of the peak location with respect tonoise in the data, a signal free of noise must be defined, and thenincremental amounts of noise is added to the signal and the impact ofthis noise on the peak location is examined. The ideal signal, forpurposes of this experiment, is the average of 10 resonant spectraacquisitions.

[0296] Gaussian noise of varying degrees is superimposed on the idealsignal. For each such manufactured noisy signal, the peak location isestimated using the 2^(nd)-order exponential curve fit. This is repeated25 times, so that the average, maximum, and minimum peak locations aretabulated. This is repeated for a wide range of noise variances—from avariance of 0 to a variance of 750. The result is shown in FIG. 47.TABLE 3 Comparison of peak location as a function of window locationShift Window Peak Location Δ = −10 bins 771.25-782.79 nm 778.8221 nm Δ =0 bins 772.70-784.23 nm 778.8887 nm Δ = +10 bins 774.15-785.65 nm7778.9653 nm

[0297] The conclusion of this experiment is that the peak locationestimation routine is extremely robust to noisy signals. The entirerange of peak locations in FIG. 47 is only 1.5 nm, even with as muchrandom noise variance of 750 superimposed—an amount of noise that issubstantially greater that what has been observed on the biosensor thusfar. The average peak location, despite the level of noise, is within0.1 nm of the ideal location.

[0298] Based on these results, a basic algorithm for mathematicallydetermining the peak location of a calorimetric resonant biosensor is asfollows:

[0299] 1. Input data x_(i)and y_(i), i=1, . . . ,n

[0300] 2. Find maximum

[0301] a. Find k such that y_(k)≧y_(i) for all i≠k

[0302] 3. Check that maximum is sufficiently high

[0303] a. Compute mean {overscore (y)} and standard deviation σ ofsample

[0304] b. Continue only if (y_(k)−{overscore (y)})/σ>UserThreshold

[0305] 4. Define curve-fit region of 2w+1 bins (w defined by the user)

[0306] a. Extract x_(i),k−w≦i≦k+w

[0307] b. Extract y_(i),k−w≦i≦k+w

[0308] 5. Curve fit

[0309] a. g_(i)=1ny_(i)

[0310] b. Perform 2^(nd)-order polynomial fit to obtain g′_(i) definedon x_(i),k−w≦i≦k+w

[0311] c. Polynomial fit returns coefficients a,b,c of form ax²+bx +c

[0312] d. Exponentiate: y′_(i)=e^(g′) ^(_(i))

[0313] 6. Output

[0314] a. Peak location p given by x_(p)=b/2a

[0315] b. Peak value given by y′_(p) (xp)

[0316] In summary, a robust peak determination routine has beendemonstrated; the statistical results indicate significant insensitivityto the noise in the signal, as well as to the windowing procedure thatis used. These results lead to the conclusion that, with reasonablenoise statistics, that the peak location can be consistently determinedin a majority of cases to within a fraction of a nm, perhaps as low as0.1 to 0.05 nm.

EXAMPLE 17

[0317] Homogenous Assay Demonstration

[0318] An SWS biosensor detects optical density of homogenous fluidsthat are in contact with its surface, and is able to differentiatefluids with refractive indices that differ by as little as n=4×10⁻⁵.Because a solution containing two free non-interacting proteins has arefractive index that is different from a solution containing two boundinteracting proteins, an SWS biosensor can measure when aprotein-protein interaction has occurred in solution without any kind ofparticle tag or chemical label.

[0319] Three test solutions were prepared for comparison:

[0320] 1. Avidin in Phosphate Buffer Solution (PBS), (10 g/ml)

[0321] 2. Avidin (10 g/ml)+Bovine Serum Albumin (BSA) (10 g/ml) in PBS

[0322] 3. Avidin (10 g/ml)+Biotinylated BSA (b-BSA) (10 g/ml) in PBS

[0323] A single SWS sensor was used for all measurements to eliminateany possibility of cross-sensor bias. A 200 1 sample of each testsolution was applied to the biosensor and allowed to equilibrate for 10minutes before measurement of the SWS biosensor peak resonant wavelengthvalue. Between samples, the biosensor was thoroughly washed with PBS.

[0324] The peak resonant wavelength values for the test solutions areplotted in FIG. 51. The avidin solution was taken as the baselinereference for comparison to the Avidin+BSA and Avidin+b-BSA solutions.Addition of BSA to avidin results in only a small resonant wavelengthincrease, as the two proteins are not expected to interact. However,because biotin and avidin bind strongly (Kd=10⁻¹⁵M), the avidin+b-BSAsolution will contain larger bound protein complexes. The peak resonantwavelength value of the avidin+b-BSA solution thus provides a largeshift compared to avidin+BSA.

[0325] The difference in molecular weight between BSA (MW—66 KDa) andb-BSA (MW—68 KDa) is extremely small. Therefore, the differencesmeasured between a solution containing non-interacting proteins(avidin+BSA) and interacting proteins (avidin+b-BSA) are attributableonly to differences in binding interaction between the two molecules.The bound molecular complex results in a solution with a differentoptical refractive index than the solution without bound complex. Theoptical refractive index change is measured by the SWS biosensor.

We claim:
 1. A biosensor comprising: (a) a two-dimensional gratingcomprised of a material having a high refractive index; (b) a substratelayer that supports the two-dimensional grating; and (c) one or morespecific binding substances immobilized on the surface of thetwo-dimensional grating opposite of the substrate layer; wherein, whenthe biosensor is illuminated a resonant grating effect is produced onthe reflected radiation spectrum, and wherein the depth and period ofthe two-dimensional grating are less than the wavelength of the resonantgrating effect.
 2. The biosensor of claim 1, wherein a narrow band ofoptical wavelengths is reflected from the biosensor when the biosensoris illuminated with a broad band of optical wavelengths.
 3. Thebiosensor of claim 1, wherein the substrate comprises glass, plastic orepoxy.
 4. The biosensor of claim 1, wherein the two-dimensional gratingis comprised of a material selected from the group consisting of zincsulfide, titanium dioxide, tantalum oxide, and silicon nitride.
 5. Thebiosensor of claim 1, further comprising a cover layer on the surface ofthe two-dimensional grating opposite of the substrate layer, wherein theone or more specific binding substances are immobilized on the surfaceof the cover layer opposite of the two-dimensional grating.
 6. Thebiosensor of claim 5, wherein the cover layer comprises a material thathas a lower refractive index than zinc sulfide, titanium dioxide,tantalum oxide, and silicon nitride.
 7. The biosensor of claim 6,wherein the cover layer comprises a material selected from the groupconsisting of glass, epoxy, and plastic.
 8. The biosensor of claim 1,wherein the two-dimensional grating has a period of about 0.01 micronsto about 1 micron and a depth of about 0.01 microns to about 1 micron.9. The biosensor of claim 1, wherein the one or more specific bindingsubstances are arranged in an array of distinct locations.
 10. Thebiosensor of claim 1, wherein the one or more specific bindingsubstances are immobilized on the two-dimensional grating by physicaladsorption or by chemical binding.
 11. The biosensor of claim 9, whereinthe distinct locations define a microarray spot of about 50-500 micronsin diameter.
 12. The biosensor of claim 1, wherein the one or morespecific binding substances are bound to their binding partners.
 13. Thebiosensor of claim 1, wherein the one or more specific bindingsubstances are selected from the group consisting of nucleic acids,polypeptides, antigens, polyclonal antibodies, monoclonal antibodies,single chain antibodies (scFv), F(ab) fragments, F(ab′)₂ fragments, Fvfragments, small organic molecules, cells, viruses, bacteria, andbiological samples.
 14. The biosensor of claim 13, wherein thebiological sample is selected from the group consisting of blood,plasma, serum, gastrointestinal secretions, homogenates of tissues ortumors, synovial fluid, feces, saliva, sputum, cyst fluid, amnioticfluid, cerebrospinal fluid, peritoneal fluid, lung lavage fluid, semen,lymphatic fluid, tears, and prostatitc fluid.
 15. The biosensor of claim12, wherein the binding partners are selected from the group consistingof nucleic acids, polypeptides, antigens, polyclonal antibodies,monoclonal antibodies, single chain antibodies (scFv), F(ab) fragments,F(ab′)₂ fragments, Fv fragments, small organic molecules, cells,viruses, bacteria, and biological samples.
 16. The biosensor of claim15, wherein the biological sample is selected from the group consistingof blood, plasma, serum, gastrointestinal secretions, homogenates oftissues or tumors, synovial fluid, feces, saliva, sputum, cyst fluid,amniotic fluid, cerebrospinal fluid, peritoneal fluid, lung lavagefluid, semen, lymphatic fluid, tears, and prostatitc fluid.
 17. Aliquid-containing vessel comprising the biosensor of claim 1 as aninternal surface.
 18. The liquid-containing vessel of claim 17, whereinthe vessel is selected from the group consisting of a microtiter plate,a test tube, a petri dish and a microfluidic channel.
 19. A detectionsystem comprising the biosensor of claim 1, a light source that directslight to the biosensor, and a detector that detects light reflected fromthe biosensor, wherein a polarizing filter occurs between the lightsource and the biosensor.
 20. A method of detecting the binding of oneor more specific binding substances to their respective binding partnerscomprising: (a) applying one or more binding partners to the biosensorof claim 1; (b) illuminating the biosensor with light; and (c) detectinga maxima in reflected wavelength, or a minima in transmitted wavelengthof light from the biosensor; wherein, if the one or more specificbinding substances have bound to their respective binding partners, thenthe reflected wavelength of light is shifted.
 21. A method of detectingthe binding of one or more specific binding substances to theirrespective binding partners comprising: (a) applying one or more bindingpartners to the biosensor of claim 1, wherein the two-dimensionalgrating is coated with an array of distinct locations containing the oneor more specific binding substances; (b) illuminating each distinctlocation of the biosensor with light; and (c) detecting maximumreflected wavelength or minimum transmitted wavelength of light fromeach distinct location of the biosensor; wherein, if the one or morespecific binding substances have bound to their respective bindingpartners at a distinct location, then the reflected wavelength of lightis shifted.
 22. A method of detecting activity of an enzyme comprising:(a) applying one or more enzymes to the biosensor of claim 1; (b)washing the biosensor; (c) illuminating the biosensor with light; and(d) detecting reflected wavelength of light from the biosensor; wherein,if the one or more enzymes have altered the one or more specific bindingsubstances of the biosensor by enzymatic activity, then the reflectedwavelength of light is shifted.
 23. A biosensor comprising: (a) a sheetmaterial having a first and second surface, wherein the first surfacedefines relief volume diffraction structures; (b) a reflective materialcoated onto the first surface of the sheet material; and (c) one or morespecific binding substances immobilized on the reflective material;wherein the biosensor reflects light predominantly at a first singleoptical wavelength when illuminated with a broad band of opticalwavelengths, and wherein the biosensor reflects light at a second singleoptical wavelength when the one or more specific binding substances areimmobilized on the reflective surface, wherein the reflection at thesecond optical wavelength of light results from optical interference.24. The biosensor of claim 23, wherein the biosensor reflects light at athird single optical wavelength when the one or more specific bindingsubstances are bound to their respective binding partners, wherein thereflection at the third optical wavelength results from opticalinterference.
 25. The biosensor of claim 23, wherein the depth andperiod of the relief volume diffraction structures are less than theresonance wavelength of the light reflected from the biosensor.
 26. Thebiosensor of claim 23, wherein the relief volume diffraction structureshave a period of about 0.01 microns to about 1 micron and a depth ofabout 0.01 micron to about 1 micron.
 27. The biosensor of claim 23,wherein the one or more specific binding substances are bound to theirrespective binding partners.
 28. A liquid-containing vessel comprisingthe biosensor of claim 23 as an internal surface.
 29. Theliquid-containing vessel of claim 28, wherein the vessel is selectedfrom the group consisting of a microtiter plate, a test tube, a petridish and a microfluidic channel.
 30. The biosensor of claim 23, whereinthe one or more specific binding substances are arranged in an array ofdistinct locations on the reflective material.
 31. The biosensor ofclaim 30, wherein the distinct locations define a microarray spot ofabout 50-500 microns in diameter.
 32. The biosensor of claim 23, whereinthe one or more specific binding substances are immobilized to thereflective material by physical adsorption or by chemical binding. 33.The biosensor of claim 32, wherein the relief volume diffractionstructures are about 0.5 microns to about 5 microns in diameter.
 34. Amethod of detecting the binding of one or more specific bindingsubstances to their respective binding partners comprising: (a) applyingone or more binding partners to the biosensor of claim 23; (b)illuminating the biosensor with light; and (c) detecting reflectedwavelength of light from the biosensor; wherein, if the one or morespecific binding substances have bound to their respective bindingpartners, then the reflected wavelength of light is shifted.
 35. Amethod of detecting the binding of one or more specific bindingsubstances to their respective binding partners comprising: (a) applyingone or more binding partners to the biosensor of claim 23, wherein theone or more specific binding substances are arranged in an array ofdistinct locations on the reflective material; (b) illuminating eachdistinct location of the biosensor with light; and (c) detectingreflected wavelength of light from each distinct location of thebiosensor; wherein, if the one or more specific binding substances havebound to their respective binding partners at a distinct location, thenthe reflected wavelength of light is shifted.
 36. A method of detectingactivity of an enzyme comprising: (a) applying one or more enzymes tothe biosensor of claim 23, (b) washing the biosensor; (c) illuminatingthe biosensor with light; and (d) detecting reflected wavelength oflight from the biosensor; wherein, if the one or more enzymes havealtered the one or more specific binding substances of the biosensor byenzymatic activity, then the reflected wavelength of light is shifted.37. A biosensor comprising a two-dimensional grating having a first anda second surface comprised of an optically transparent material thatconducts electricity, wherein the first surface of the two-dimensionalgrating is coated with an electrical insulator, and wherein the secondsurface of the two-dimensional grating is deposited on a substrate,wherein when the biosensor is illuminated a resonant grating effect isproduced on the reflected radiation spectrum, wherein the depth and theperiod of the two-dimensional grating are less than the wavelength ofthe resonant grating effect.
 38. The biosensor of claim 37, wherein thetwo-dimensional grating is comprised of a repeating pattern of shapesselected from the group consisting of squares, circles, ellipses,triangles, ovals, trapezoids, sinusoidal waves, rectangles, andhexagons.
 39. The biosensor of claim 37, wherein the repeating patternof shapes are arranged in a rectangular grid or hexagonal grid.
 40. Thebiosensor of claim 37, wherein the two-dimensional grating has a periodof about 0.01 microns to about 1 micron and a depth of about 0.01microns to about 1 micron.
 41. The biosensor of claim 37, wherein two ormore separate grating regions are present on the same substrate.
 42. Thebiosensor of claim 41, further comprising an electrically conductingtrace to each separate grating region of the substrate.
 43. Thebiosensor of claim 42, wherein the conducting trace is connected to avoltage source.
 44. The biosensor of claim 41, wherein one or morespecific binding substances are bound to each separate grating region ofthe substrate.
 45. The biosensor of claim 44, wherein the one or morespecific binding substances are bound to their respective bindingpartners.
 46. A liquid-containing vessel comprising the biosensor ofclaim 37 as an internal surface.
 47. The liquid-containing vessel ofclaim 46, wherein the vessel is selected from the group consisting of amicrotiter plate, a test tube, a petri dish and a microfluidic channel.48. A method of detecting the binding of one or more specific bindingsubstances to their respective binding partners comprising: (a) applyingone or more binding partners to the biosensor of claim 37; (b) applyingan electrical charge to the electrically conducting traces; (c)illuminating the biosensor with light; and (d) detecting reflectedwavelength of light from the biosensor; wherein, if the one or morespecific binding substances have bound to their respective bindingpartners, then the reflected wavelength of light is shifted.
 49. Themethod of claim 48, further comprising the step of applying a reversedelectrical charge to the electrically conducting traces beforeilluminating the biosensor with light.
 50. A method of measuring theamount of one or more binding partners in a test sample comprising: (a)illuminating the biosensor of claims 1, 23, or 37 with light; (b)detecting reflected wavelength of light from the biosensor; (d) applyinga test sample comprising one or more binding partners to the biosensor;(e) illuminating the biosensor with light; and (f) detecting reflectedwavelength of light from the biosensor; wherein, the difference inwavelength of light in step (b) and step (f) is a measurement of theamount of one or more binding partners in the test sample.
 51. Adetection system comprising the biosensor of claims 1, 23, or 37, alight source that directs light at the biosensor, and a detector thatdetects light reflected from the biosensor, wherein a first illuminatingfiber probe having two ends is connected at its first end to thedetector, wherein a second collection fiber probe having two ends isconnected at its first end to the light source, wherein the first andsecond fiber probes are connected at their second ends to a third fiberprobe, wherein the third fiber probe acts as an illumination andcollection fiber probe, and wherein the third fiber probe is oriented ata normal angle of incidence to the biosensor and supportscounter-propagating illuminating and reflecting optical signals.
 52. Adetection system comprising the biosensor of claims 1, 23, or 37, alight source that directs light at the biosensor, and a detector thatdetects light reflected from the biosensor, wherein an illuminatingfiber probe is connected to the light source and is oriented at a 90degree angle to a collecting fiber probe, wherein the collecting fiberprobe is connected to the detector, wherein light is directed throughthe illuminating fiber probe into a beam splitter that directs the lightto the biosensor, wherein reflected light is directed into the beamsplitter that directs the light into the collecting fiber.
 53. A methodof immobilizing one or more specific binding substances onto thebiosensor of claim 1, 23, or 37 comprising activating the biosensor withamine, attaching linker groups to the amine-activated biosensor, andattaching one or more specific binding substances to the linker groups.54. The method of claim 53, wherein the biosensor is activated withamine by a method comprising: (a) immersing the biosensor into a piranhasolution; (b) washing the biosensor; (c) immersing the biosensor in 3%3-aminopropyltriethoxysilane solution in dry acetone; (d) washing thebiosensor in dry acetone; and (e) washing the biosensor with water. 55.The method of claim 53, wherein the linker is selected from the groupconsisting of amine; aldehyde; N,N′-disuccinimidyl carbonate; andnickel.
 56. A method of detecting the binding of one or more specificbinding substances to their respective binding partners comprising: (a)applying one or more binding partners comprising one or more tags to thebiosensor of claims 1, 23, or 37; (b) illuminating the biosensor withlight; and (c) detecting reflected wavelength of light from thebiosensor; wherein, if the one or more specific binding substances havebound to their respective binding partners, then the reflectedwavelength of light is shifted.
 57. The method of claim 56, wherein theone or more tags are selected from the group consisting of biotin,succinimidyl-6-[a-methyl-a-(2-pyridyl-dithio) toluamido] hexanoate(SMPT), dimethylpimelimidate (DMP), and histidine.
 58. The method ofclaim 56, wherein the one or more tags are reacted with a compositionselected from the group consisting of streptavidin, horseradishperoxidase, and streptavidin coated nanoparticles, before the step ofilluminating the biosensor with light.
 59. A biosensor compositioncomprising two or more biosensors of claims 9, 30, or 44 wherein thebiosensors are associated with a holding fixture.
 60. The biosensorcomposition of claim 59, wherein the composition comprises about 50 toabout 1,000 individual biosensors.
 61. The biosensor composition ofclaim 59, wherein the composition comprises about 96 biosensors.
 62. Thebiosensor composition of claim 59, wherein the composition comprisesabout 384 biosensors.
 63. The biosensor composition of claim 59, whereinthe two or more biosensors each comprise about 25 to about 1,000distinct locations.
 64. The biosensor composition of claim 59, whereineach biosensor is about 1 mm² to about 5 mm².
 65. The biosensorcomposition of claim 59, wherein each biosensor is about 3 mm².
 66. Thebiosensor composition of claim 59, wherein the holding fixture holdseach biosensor such that each biosensor can be placed into a separatewell of a microtiter plate.
 67. A biosensor composition comprising oneor more biosensors of claims 1, 23, or 37 on a tip of a multi-fiberoptic probe.
 68. The biosensor composition of claim 67, wherein the oneor more biosensors are fabricated into the tip of the probe.
 69. Thebiosensor composition of claim 67, wherein the one or more biosensorsare attached onto the tip of the probe.
 70. A method of detectingbinding of one or more specific binding substances to their respectivebinding partners in vivo comprising: (a) inserting the tip of the fiberoptic probe of claim 67 into the body of a human or animal; (b)illuminating the biosensor with light; (c) detecting reflectedwavelength of light from the biosensor; wherein, if the one or morespecific binding substances have bound to their respective bindingpartners, then the reflected wavelength of light is shifted.
 71. Adetection system comprising: (a) the biosensor of claim 1; (b) a lasersource that directs a laser beam to a scanning mirror device, whereinthe scanning mirror device is used to vary the laser beam's incidentangle; (c) an optical system for maintaining columination of theincident laser beam; (d) and a light detector.
 72. The detection systemof claim 71, wherein the scanning mirror device is a lineargalvanometer.
 73. The detection system of claim 72, wherein the lineargalvanometer operates at a frequency of about 2 Hz to about 120 Hz and amechanical scan angle of about 10 degrees to about 20 degrees.
 74. Thedetection system of claim 71, wherein the laser is a diode laser with awavelength selected from the group consisting of 780 nm, 785 nm, 810 nm,and 830 nm.
 75. A method for determining a location of a resonant peakfor a binding partner in a resonant reflectance spectrum with acolormetric resonant biosensor, comprising: selecting a set of resonantreflectance data for a plurality of colormetric resonant biosensordistinct locations, wherein the set of resonant reflectance data iscollected by illuminating a colormetric resonant diffractive gratingsurface with a light source and measuring reflected light at apredetermined incidence, wherein the colormetric resonant diffractivegrating surface is used as a surface binding platform for one or morespecific binding substances, wherein binding partners can be detectedwithout use of a molecular label, wherein the set of resonantreflectance data includes a plurality of sets of two measurements, wherea first measurement includes a first reflectance spectra of one or morespecific binding substances that are attached to the colormetricresonant diffractive grating surface and a second measurement includes asecond reflectance spectra of the one or more specific binding substanceafter one or more binding partners are applied to colormetric resonantdiffractive grating surface including the one or more specific bindingsubstances, and wherein a difference in a peak wavelength between thefirst and second measurement is a measurement of an amount of bindingpartners that bound to the one or more specific binding substances;determining a maximum value for a second measurement from the pluralityof sets of two measurements from the set of resonant reflectance datafor the plurality of binding partners, wherein the maximum valueincludes inherent noise included in the resonant reflectance data;determining whether the maximum value is greater than a predeterminedthreshold, and if so, defining a curve-fit region around the determinedmaximum value, performing a curve-fitting procedure to fit a curvearound the curve-fit region, wherein the curve-fitting procedure removesa pre-determined amount of inherent noise included in the resonantreflectance data; determining a location of a maximum resonant peak onthe fitted curve; and determining a value of the maximum resonant peak,wherein the value of the maximum resonant peak is used to identify anamount of biomolecular binding of the one or more specific bindingsubstances to the one or more binding partners.
 76. A computer readablemedium having stored therein instructions for causing a processor toexecute the method of claim
 75. 77. The method of claim 75 wherein asensitivity of a colormetric resonant biosensor is determined by a shiftin a location of a resonant peak in the plurality of sets of twomeasurements in the set of resonant reflectance data.
 78. The method ofclaim 75 wherein the step of selecting a set of resonant reflectancedata includes selecting a set of resonant reflectance data: x _(i) and y_(i) for i=1, 2, 3, . . . n, wherein x_(i) is where a first measurementincludes a first reflectance spectra of one or more specific bindingsubstance attached to the colormetric resonant diffractive gratingsurface, y_(i) a second measurement includes a second reflectancespectra of the one or more specific binding substances after a pluralityof binding partners are applied to colormetric resonant diffractivegrating surface including the one or more specific binding substances,and n is a total number of measurements collected.
 79. The method ofclaim 75 wherein the step of determining a maximum value for a secondmeasurement includes determining a maximum value y_(k) such that:(y_(k) >=y _(i)) for all i≠k.
 80. The method of claim 75 wherein thestep of determining whether the maximum value is greater than apre-determined threshold includes: computing a mean of the set ofresonant reflectance data; computing a standard deviation of the set ofresonant reflectance data; and determining whether((y_(k)−mean)/standard deviation) is greater than a pre-determinedthreshold.
 81. The method of claim 75 wherein the step of defining acurve-fit region around the determined maximum value includes: defininga curve-fit region of (2w+1) bins, wherein w is a pre-determinedaccuracy value; extracting (x_(i), k−w<=i<=k+w); and extracting (y_(i),k−w<=i<=k+w).
 82. The method of claim 75 wherein the step of performinga curve-fitting procedure includes: computing g_(i)=1n y_(i); performinga 2^(nd) order polynomial fit on g_(i) to obtain g′_(i) defined on(x_(i), k−w<=i<=k+w); determining from the 2^(nd) order polynomial fitcoefficients a, b and c of for (ax²+bx+c)−; and computingy′_(i)=e^(g′i).
 83. The method of claim 75 wherein the step ofdetermining a location of a maximum resonant peak on the fitted curveincludes: determining location of maximum resonant peak (x_(p)=(−b)/2a).84. The method of claim 75, wherein the step of determining a value ofthe maximum resonant peak includes determining the value with of x_(p)at y′_(p).
 85. A biosensor comprising a two-dimensional grating having apattern of concentric rings, wherein the difference between an insidediameter and an outside diameter of each concentric ring is equal toabout one-half of a grating period, wherein each successive ring has aninside diameter that is about one grating period greater than an insidediameter of a previous ring wherein when the structure is illuminatedwith an illuminating light beam, a reflected radiation spectrum isproduced that is independent of an illumination polarization angle ofthe illuminating light beam, and wherein one or more specific bindingsubstances are immobilized on the two-dimensional grating.
 86. Thebiosensor of claim 85, wherein when the structure is illuminated aresonant grating effect is produced on the reflected radiation spectrum,wherein the depth and period of the two-dimensional grating are lessthan the wavelength of the resonant grating effect, and wherein a narrowband of optical wavelengths is reflected from the structure when thestructure is illuminated with a broadband of optical wavelengths. 87.The biosensor of claim 85, wherein the two-dimensional grating has aperiod of about 0.01 microns to about 1 micron and a depth of about 0.01microns to about 1 micron.
 88. A biosensor comprising an array of holesor posts arranged such that the holes or posts are centered on cornersand in the center of hexagons, wherein the hexagons are arranged in aclosely packed array, wherein when the structure is illuminated with anilluminating light beam, a reflected radiation spectrum is produced thatis independent of an illumination polarization angle of the illuminatinglight beam, and wherein one or more specific binding substances areimmobilized on the array of holes or posts.
 89. The biosensor of claim88, wherein when the structure is illuminated a resonant grating effectis produced on the reflected radiation spectrum, wherein the depth orheight and period of the holes or posts are less than the wavelength ofthe resonant grating effect, and wherein a narrow band of opticalwavelengths is reflected from the structure when the structure isilluminated with a broad band of optical wavelengths.
 90. The structureof claim 88, wherein the array holes or posts have a period of about0.01 microns to about 1 micron and a depth of height of about 0.01microns to about 1 micron.
 91. A biosensor comprising: (a) a firsttwo-dimensional grating comprising a high refractive index material andhaving a top surface and a bottom surface; (b) a second two-dimensionalgrating comprising a high refractive index material and having a topsurface and a bottom surface, wherein the top surface of the secondtwo-dimensional grating is attached to the bottom surface of the firsttwo-dimensional grating; and (c) one or more specific binding substancesor one or more specific binding substances bound to their bindingpartners immobilized on the top surface of the first two-dimensionalgrating; wherein, when the biosensor is illuminated two resonant gratingeffects are produced on the reflected radiation spectrum, and whereinthe depth and period of both of the two-dimensional gratings are lessthan the wavelength of the resonant grating effects.
 92. The biosensorof claim 91, wherein a substrate layer supports the bottom surface ofthe second two-dimensional grating.
 93. The biosensor of claim 91,further comprising a cover layer on the top surface of the firsttwo-dimensional grating, wherein the one or more specific bindingsubstances are immobilized on the surface of the cover layer opposite ofthe two-dimensional grating.
 94. The biosensor of claim 91, wherein thetop surface of the first two-dimensional grating is in physical contactwith a test sample, and the second two dimensional grating is not inphysical contact with the test sample.
 95. The biosensor of claim 91,wherein when a peak resonant reflection wavelength is measured for thefirst and second two-dimensional gratings, the difference between thetwo measurements indicates the amount of one or more specific bindingsubstances, binding partners, or both deposited on the surface of thefirst two-dimensional grating.
 96. A biosensor comprising: (a) a firsttwo-dimensional grating comprising a high refractive index material andhaving a top surface and a bottom surface; (b) a substrate layercomprising a top surface and a bottom surface, wherein the top surfaceof the substrate supports the bottom surface of the firsttwo-dimensional grating; (c) a second two-dimensional grating comprisinga high refractive index material and having a top surface and a bottomsurface, wherein the bottom surface of the second two-dimensionalgrating is attached to the bottom surface of the substrate; and (d) oneor more specific binding substances or one or more specific bindingsubstances bound to their binding partners immobilized on the topsurface of the first two-dimensional grating; wherein, when thebiosensor is illuminated two resonant grating effects are produced onthe reflected radiation spectrum, and wherein the depth and period ofboth of the two-dimensional gratings are less than the wavelength of theresonant grating effects.
 97. The biosensor of claim 96, furthercomprising a cover layer on the top surface of the first two-dimensionalgrating, wherein the one or more specific binding substances areimmobilized on the surface of the cover layer opposite of thetwo-dimensional grating.
 98. The biosensor of claim 96, wherein the topsurface of the first two-dimensional grating is in physical contact witha test sample, and the second two dimensional grating is not in physicalcontact with the test sample.
 99. The biosensor of claim 98, whereinwhen a peak resonant reflection wavelength is measured for the first andsecond two-dimensional gratings, the difference between the twomeasurements indicates the amount of one or more specific bindingsubstances, binding partners, or both deposited on the surface of thefirst two-dimensional grating.
 100. The biosensor of claim 1, furthercomprising an antireflective dielectric coating on a surface of thesubstrate opposite of the two-dimensional grating.
 101. The biosensor ofclaim 1, wherein the biosensor is attached to a bottomless microtiterplate by a method selected from the group consisting of adhesiveattachment, ultrasonic welding and laser welding.
 102. A method ofdetecting an interaction of a first molecule with a second test moleculecomprising: (a) applying a mixture of the first and second molecules toa distinct location on a biosensor, wherein the biosensor comprises atwo-dimensional grating comprising of a high refractive index material,and a substrate layer that supports the two-dimensional grating; andwherein, when the biosensor is illuminated a resonant grating effect isproduced on the reflected radiation spectrum, and wherein the depth andperiod of the two-dimensional grating are less than the wavelength ofthe resonant grating effect; (b) applying a mixture of the firstmolecule with a third control molecule to a distinct location on thebiosensor of (a) or a similar biosensor, wherein the third controlmolecule does not interact with the first molecule, and wherein thethird control molecule is about the same size as the first molecule; and(c) detecting a shift in the reflected wavelength of light from thedistinct locations of step (a) and step (b); wherein, if the shift inthe reflected wavelength of light from the distinct location of step (a)is greater than the shift in the reflected wavelength in step (b), thenthe first molecule and the second test molecule interact.
 103. Themethod of claim 102, wherein the first molecule is selected from thegroup consisting of a nucleic acid, polypeptide, antigen, polyclonalantibody, monoclonal antibody, single chain antibody (scFv), F(ab)fragment, F(ab′)₂ fragment, Fv fragment, small organic molecule, cell,virus, and bacteria.
 104. The method of claim 102, wherein the secondtest molecule is selected from the group consisting of a nucleic acid,polypeptide, antigen, polyclonal antibody, monoclonal antibody, singlechain antibody (scFv), F(ab) fragment, F(ab′)₂ fragment, Fv fragment,small organic molecule, cell, virus, and bacteria.